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Study of Lactobacillus as Probiotic Bacteria

July 2004
Iranian Journal of Public Health 33(2)

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CC BY-NC 4.0

Authors:

Jamileh Nowroozi

Islamic Azad University

Mehdi Mirzaii

Shahroud University of Medical Sciences

Milad Norouzi

Shahrood University of Technology

Because of inhibitory effect, selected probiotic lactobacilli may be used as biological preservative, so, the aim of this study was to present some data on lactobacillus as probiotic bacteria. Lactic acid bacteria were isolated from sausage. Each isolate of lactobacillus species was identified by biochemical tests and comparing their sugar fermentation pattern. Antibacterial activities were done by an agar spot, well diffusion and blank disk method. Enzyme sensitivity of supernatant fluid and concentrated cell free culture after treatment with α-amylase, lysozyme and trypsin was determined. The isolated bacteria were Lacto. plantarum, Lacto delbruekii, Lacto. acidophilus, Lacto. brevis. The isolated bacteria had strong activity against indicator strains. The antibacterial activity was stable at 100ºC for 10 min and at 56ºC for 30 min, but activity was lost after autoclaving. The maximum production of plantaricin was obtained at 25 - 30ºC at pH 6.5. Because, lactobacilli that used to process sausage fermentation are producing antimicrobial activity with heat stability bacteriocin, so, these bacteria may be considered to be a healthy probiotic diet. Lactobacilli originally isolated from meat products are the best condidates as probiotic bacteria to improve the microbiological safety of these foods.

: Zone inhibition of Staph. aureus against supernatant of Lacto. plantarum by agar spot method. a: Lacto. Plantarum, b: Lacto. delbruekii, c: Lacto. acidophilus, d: Lacto. brevis, e: Lacto. casei

…

Zone inhibition of Staph. aureus against supernatant of Lacto. plantarum by blank disk method. a: Lacto. plantarum, b: Lacto. delbruekii, c: Lacto. acidophilus, d: Lacto. brevis, e: Lacto. casei

…

: Zone inhibition of Staph. aureus against supernatant of Lacto. plantarum by agar well diffusion method. a: Lacto. plantarum, b: Lacto. delbruekii, c: Lacto. acidophilus, d: Lacto. brevis, e: Lacto. casei

…

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Iranian J Publ Health, Vol. 33, No. 2, pp.1-7, 2004

Study of Lactobacillus as Probiotic Bacteria

*J Nowroozi

, M Mirzaii

, M Norouzi

Dept. of Medical Microbiology, Medical School, Iran University of Medical Sciences, Tehran, Iran

Dept. of Medical Virology, Medical School, Iran University of Medical Sciences, Tehran, Iran

Abstract

Because of inhibitory effect, selected probiotic lactobacilli may be used as biological preservative, so, the aim of this study

was to present some data on lactobacillus as probiotic bacteria. Lactic acid bacteria were isolated from sausage. Each isolate

of lactobacillus species was identified by biochemical tests and comparing their sugar fermentation pattern. Antibacterial

activities were done by an agar spot, well diffusion and blank disk method. Enzyme sensitivity of supernatant fluid and

concentrated cell free culture after treatment with α-amylase, lysozyme and trypsin was determined. The isolated bacteria

were Lacto. plantarum, Lacto delbruekii, Lacto. acidophilus, Lacto. brevis. The isolated bacteria had strong activity against

indicator strains. The antibacterial activity was stable at 100ºC for 10 min and at 56ºC for 30 min, but activity was lost after

autoclaving. The maximum production of plantaricin was obtained at 25 - 30ºC at pH 6.5. Because, lactobacilli that used to

process sausage fermentation are producing antimicrobial activity with heat stability bacteriocin, so, these bacteria may be

considered to be a healthy probiotic diet. Lactobacilli originally isolated from meat products are the best condidates as

probiotic bacteria to improve the microbiological safety of these foods.

Keywords: Lactobacillus, Probiotic, Sausage, Antibacterial activity, Iran

Introduction

Probiotic cultures have been associated histori-

cally with cultured of milks and dairy products,

from which there is substantial evidence for

positive effects on human health and general

well-being (1, 2). Several in vitro and in vivo

experiments on antagonism of different Lacto-

bacillus strains against Helicobacter pylori and

Clostridium difficile, Campylobacter jejuni, E.

coli were performed. All tested human Lacto-

bacillus strains were able to inhibit the growth

of all strains of anaerobic human gastrointesti-

nal pathogens (3, 4). In addition, bacteriocins

have properties such as antitumour and anticho-

lestrol activity. Chemical reactions associated

with reduction of nitrate, improvements inimmu

nological status and adsorption of vitamins B

group (5). The transity lactic acid bacteria in

the gastrointestinal tract are capable of deliver-

ing enzymes and other substances into the

intestine which possibly help to control intesti-

nal flora (6). Also, the antioxidative activity of

lactic acid bacteria is reported (7). Because of

inhibitory effect, selected probiotic lactobacilli

may be used as biological preservative, so, the

aim of this study was to present some data on

isolation, growth, and antimicrobial activity,

effect of pH, heat, and sensitivity to proteolytic

enzymes of lactobacillus as probiotic bacteria.

Materials and Methods

Each of the following experiments was repeated

thrice to get better results.

Isolation The isolation of lactic acid bacteria

from sausages bought randomly from shops

with different brand of factory was done by

using MRS (pepton, meat extract, yeast extract,

glucose, tween 80) medium. Briefly, 1g of sau-

sage was mixed and vortexed into MRS broth

medium, incubated at 37ºC for 24 h. Growth

from MRS broth cultures was used to streak on

MRS agar plate. Lactobacillus species of these

Iranian J Publ Health, Vol. 33, No. 2,

.1 -7

200

*Corresponding author: P. O. BOX: 14155-6183, Fax: +98 21 8058719

J Nowroozi et al: Lactobacillus as…

isolates were identified by comparing their

sugar fermentation patterns with the scheme de-

scribed in Bergey’s Manual of Systematic

Bacteriology (8).

Growth of bacteria Lactobacilli were grown

in MRS broth or MRS agar. One ml of an

overnight culture of lactobacillus was used to

inoculate 100 ml of MRS broth and incubation

was continued at 20, 25, 30, 35, 37, 40, 45ºC

for 24 h. Samples were removed at regular

intervals (30 min) for the determination of

turbidity (measured at 660 nm), culture pH and

antibacterial activity. The experiment was re-

peated with broth in which the initial pH was

adjusted to 2 to 12 with HCl or NaCl. Initial

and final pH of all samples was also measured.

Culture supernatant (200µl) was heated in a

boiling water bath for 10 min and cooled rap-

idly on ice. Serial twofold dilutions of the

heated supernatants were made in 0.2 N HCl,

and 10µl of each dilution was spotted on to

fresh, duplicate indicator lawns. Cultures were

incubated for 24 h.

Effect of different sugar and NaCl concentra-

tion on production of bacteriocin Isolated

lactobacillus was grown in MRS broth without

beef extract, supplemented with different con-

centration of glucose, xylose, sucrose, furoc-

tose, galactose, maltose and NaCl. Then, re-

maining activity against indicator strains was

assayed.

Preparation of culture supernatant The

bacteriocin producing strain was grown in MRS

broth for 24 h at 25ºC. A cell free solution was

obtained by centrifuge the culture, followed by

filtration of the supernatant through a 0.2 µl

pore size filter. The supernatant was adjusted to

pH 6.5 or dialyzed for 24 h against MRS broth

at 4ºC.

Mode of action one ml of cell free culture

supernatant of isolated lactobacilli was added to

10 ml of a fresh culture logarithmic phase of

indicator bacteria. Culture optical density were

determined (at 660 nm) at appropriate intervals.

Antimicrobial activity For detection of

antagonistic activities, an agar spot procedure,

well diffusion assay and blank disk method

were used. For the agar spot test, supernatant of

overnight cultures of lactobacillus strains were

spotted (1mm) onto the surface of BHI agar

plates of indicator strains and incubated for 24

h at 37ºC to allow colony develop. For the agar-

well diffusion assay, an overnight culture of the

indicator strain was used to inoculate agar

growth media at 37ºC. Wells of 5mm diameter

were cut into agar plates and 50 µl of culture

supernatant fluid containing antibacterial activ-

ity were added to each well. Supernatant fluid

was obtained by growing the inhibitory pro-

ducer strain overnight in MRS broth at 30ºC.

Cells were then removed by centrifugation and

the supernatant fluid placed in the wells and

allowed to diffuse into the agar for 24 h at 4ºC.

The plates were then incubated at optimum

growth temperature of the indicator strains and

examined after 24 h for inhibition zone. Five

sterile paper blank disks were placed on the

agar plate which was inoculated by indicator

strains and 20 µl of the filtered supernatant of

lactobacilli was applied. Plates were incubated

and observed for zones inhibition.

Indicator strains used as indicator organisms for

bacteriocin screening were Staphylococcus

aureus, Salmonella typhi, Yersinia enterocoli-

tica, Bacillus subtilis, Listeria monocytogenes

and other lactobacilli isolated from sausage

without antibacterial activity. The plates were

incubated at 30ºC for 24-48 h or until growth of

the test organism could be easily observed with

naked eye.

Sensitivity to pH and heat To test sensitivity

to pH, the supernatant was adjusted to pHs be-

tween 2 to 12 with HCl or NaOH and incu-

bated. To test heat stability, the supernatant

fluid was heated in boiling water for 10 min, at

56ºC for 15 min, or autoclaved at 121ºC for 15

min. In all cases, the activity remaining after

treatment was measured by spotting procedure.

This experiment repeated and the solutions

were kept at 4 and -20ºC for 4 weeks, then

antibacterial activity was measured. Sensitivity

Sensitivity proteolytic enzymes To test for

Iranian J Publ Health, Vol. 33, No. 2, pp.1-7, 2004

enzyme sensitivity, cell free culture supernatant

fluid was treated for 1h at 30ºC with trypsin, α-

amylase, lysozyme at final concentration of

0.5mg/ml, 220 IU/mg/ml and 22IU/mg/ml, and

incubated at 37ºC for 1 h.Concentrated cell free

culture supernatants were heated at 100ºC for

20 min and the remaining activity was deter-

mined by spotted procedure.

Bacteriocin concentration one liter

lactobacilli culture was grown in MRS broth at

30ºC until the late logarithmic phase. The cell

removed by centrifugation for 12 min. at 4ºC,

and ammonium sulphate was gradually added

to achieve 40% saturation. The sample was kept

at 4ºC with stirring for 30 min. After centrifu-

gation for 30 min, the resulting pellet was

mixed and solubilized in 120 ml of 10 mM so-

dium phosphate buffer, pH 5.8. Then antimicro-

bial activity was measured against indicator

bacteria (9).

Results

A total of 28 lactic acid bacteria isolated from

sausage were tested for antimicrobial activity.

Only 4 strains (14.3%) of lactobacilli (Lacto.

plantarum, Lacto. delbruekii, Lacto. acidophi-

lus, Lacto. brevis) were shown to produce a

bacteriocin-like substance. Their sensitivity

varied greatly. Lacto. plantarum produced a

more heat stable bacteriocin than the other

isolated strains, which exhibited a broad spec-

trum of inhibitory activity. The antibacterial

activity of plantaricin was more potent than the

other isolated strains when sensitive strains

were in the logarithmic growth phase, including

cell lysis, as observed by decreased in optical

density. No bacteriocin activity was found in

cultures grown at 4 or 8ºC. However, bacterio-

cin production was observed at 20, 25, 30, 37,

40 and 45ºC. At all of these temperatures, the

maximum antimicrobial activity in the growth

medium was obtained in the late logarithmic

phase growth and early of stationary phase.

The amounts of bacteriocin produced at 25 and

30ºC were similar. The bacteriocin activity in

the supernatant was stable and no decrease in

activity was detected after 5 days at 25ºC. The

antibacterial activity was stable at 100ºC for 10

min and at 56ºC for 30 min, but all activity was

lost after autoclaving. The antibacterial activity

was not lost by freezing and thawing, and long

term storage at 4 and -20ºC. When the super-

natants of the cultures containing Lacto. planta-

rum were checked, a small zone of inhibition

was first observed on plates after 6 h at 25ºC

and larger zones of inhibition were detected

after 24 h.

Factors affecting bacteriocin activity The

antibacterial activity of bacteriocin was de-

stroyed by trypsin treatment, but was unaf-

fected by α-amylase and lysozyme.

The inhibitory activity remained stable over the

pH range 2 to10, but was lost after incubation at

pH 12, indicating its sensitivity to alkali treat-

ment. All activity was lost after autoclaving.

The antimicrobial properties of the Lacto-

lactobacillus strains tested were very variable.

Many of the strains showed weak or no inhibi-

tion of the pathogenic strains.

Only 4 strains (14.3%) inhibited the growth of

pathogenic bacteria broadly. The maximum

production of the bacteriocin was obtained at

25ºC at pH 6.5.

Bacteriocin production Maximum

production of bacteriocin was obtained in MRS

broth containing at least 1-2% glucose or xy-

lose. Also, MRS medium with 1% NaCl found

that, the antibacterial activity increased. The

inhibitory activity was maximal at the

beginning of the stationary phase and remained

stable long after growth had ceased, even in the

presence of the producer cells.

Zone inhibition of Staph.aureus against super-

natant of lactobacilli by agar spot method,

blank disk, and agar well diffusion assay are

shown in Figs 1, 2, 3.

J Nowroozi et al: Lactobacillus as…

Fig. 1 : Zone inhibition of Staph. aureus against supernatant of Lacto. plantarum by

agar spot method.

a: Lacto. Plantarum, b: Lacto. delbruekii, c: Lacto. acidophilus, d: Lacto. brevis, e: Lacto. casei

Fig. 2: Zone inhibition of Staph. aureus against supernatant of Lacto. plantarum by

blank disk method.

a: Lacto. plantarum, b: Lacto. delbruekii, c: Lacto. acidophilus, d: Lacto. brevis, e: Lacto. casei

Iranian J Publ Health, Vol. 33, No. 2, pp.1-7, 2004

Fig. 3 : Zone inhibition of Staph. aureus against supernatant of Lacto. plantarum by

agar well diffusion method.

a: Lacto. plantarum, b: Lacto. delbruekii, c: Lacto. acidophilus, d: Lacto. brevis, e: Lacto. casei

Discussion

Of 28 lactobacilli isolated from fermented

sausage, 4 (14.3%) lactobacilli had antibacterial

activity against indicator strains ( L. monocyto-

genes, Y. enterocolitica, Staph. aureus, B. sub-

tilis, S. typhi and other isolated lactobacilli with

no antibacterial activity), which were further

characterized. Their antimicrobial substances

inactivated by trypsin and designed as bacterio-

cin (plantaricin). Lacto. plantarum (plantaricin)

showed the broadest range of inhibitory action.

This is agreeing by the results of Kelley et al

(9) and not agrees elsewhere (10). The latter

reported Lacto. casei with a potent antimicro-

bial activity.

Since antibacterial activity decreased after

treatment with trypsin, but not affected with

lysozyme and α-amylase, so, the bacteriocins

have probably a pertinacious nature. This was

in accordance with Gonzalez et al (11). Also,

the protein nature of plantaricin K was

confirmed by its sensitivity to trypsin (12).

Bacteriocin of Lacto. plantarum on the basis of

its stability in the medium, its broad spectrum

of activity on some pathogenic and spoilage

food bacteria and its high potency of plantaricin

production is recommended as food preserva-

tive. Our results showed that bactericidal action

of the bacteriocin against indicator strains were

on logarithmic phase and early stationary phase

and cell lyses in actively growing cells, thereby

causing a decrease in culture optical density.

This was also confirmed by Gao et al (13).

In this study, production of plantaricin was best

in MRS broth, or in a medium containing

peptone, yeast extract, beef extract, glucose,

sodium acetate and Tween 80. Glucose could

be replaced by xylose without a decrease in the

amount of plantaricin, but other carbohydrates

resulted in less bacteriocin being produced.

J Nowroozi et al: Lactobacillus as…

Maximum production was coincided with onset

of logariyhmic phase and early of stationary

phase, and these conditions of low pH and high

cell number have also been found to be neces-

sary for the production of high levels of bac-

teriocins. Maximum production of plantaricin

KW30 (9) and bacteriocin of Lacto. delbrueckii

(14) were in MRS broth. Their results are simi-

lar to our results.

Our results showed that bacteriocin activity was

very stable under a series of different condi-

tions such as storage at room temperature for 5

days, 4ºC and -20ºC, and heating (100ºC for 10

min or 56ºC for 30 min). This is confirmed by

Rekhif et al (15).

In general, bacteriocin are from lactobacilli

specially Lacto. plantarum relatively heat stable

with promising inhibitory spectra of antimicro-

bial activities.

Their general heat stability is an advantage,

temperature stability being a very important

parameter if a bacteriocin is to be used as a

food preservative because many proc- essing

procedures involve a heating step.

However, the bacteriocins from Lacto. planta-

rum described in this paper appear quite prom-

ising as potential biopreservatives. Our results

are confirmed by some researchers (9, 11, 15).

Lactic acid bacteria originally isolated from

meat, meat products and dairy products are

probably the best candidates as probiotic bacte-

ria to improve the microbiological safety of

these foods. Since, they are well adapted to the

conditions in meats and dairy products and

should therefore be more competitive than from

other sources.

Interest in lactic acid bacteria is growing. Also,

bacteriocins produced by lactic acid bacteria are

great interest to the food fermentation industry

because they may inhibit the growth of many

food spoilage and pathogenic bacteria. There-

fore, an investigation of bacteriocins in lactic

acid bacteria may offer potential applicability in

food preservation.

Acknowledgements

We would like to thank to laboratory staff in

Dept. of Microbiology, Iran University of

Medical Sciences, Iran this study was a part of

Mr Mehdi Mirzaii thesis.

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Article

Aug 2022

Background: Probiotic refer to harmless live normal flora micro-organisms (M.O), which are mostly strains of Lactobacillus spp., Bifidobacterium spp., and others. These microorganisms (M.O) help the host's health when given in the right dosages. Aims: To identify bacterial species isolated from wound infections, and also to evaluate in-vitro the antimicrobial activity of selected probiotic species and antibiotic against some pathogenic bacteria. Methods: Records of wound swabs collected from 102 patients with high suspicion of wound infections. Bacterial colonies were Gram stained and microscopically examined. Biochemical tests were done to identify pathogen species. The microtitre plate method (MTP) was used to detect biofilm formation by bacteria. The Kirby-Bauer disk diffusion method was used for antibiotic testing. The well diffusion method and agar spot method was used for detection probiotic affectivity to a group of bacteria isolated (totally 38 isolates). Results: Prevalence of wound infections was 81.4% (CI: 60.869). A total of 10 species were isolated from 83 infected wounds. The most common bacterial species detected was Staphylococcus aureus (26.5%), followed by Pseudomonas aeruginosa 14.4%, Proteus mirabilis (12%), Escherichia coli 10.8%, and Enterobacter cloacae (8.4%). Polymicrobial infection was found in 25(30.1%) of the samples and was mainly constituted with two species.

Characterization of Lactiplantibacillus plantarum Tw226 strain and its use for the production of a new membrane-bound biosurfactant

Article

Jul 2022
J MOL LIQ

This study aimed to evaluate the viability for biotechnological uses of a new cell-bound biosurfactant produced by a lactic acid bacterium isolated from the intestinal tract of Odontesthes bonariensis. For this purpose, the bacterial strain was characterized, and the conditions for biosurfactant production were optimized. Moreover, the biosurfactant was chemically characterized, and its possible use as an emulsifying agent assessed. The bacterium was identified as Lactiplantibacillus plantarum Tw226 and qualified as safe for biotechnological uses according to the genomic and antibiotic resistance studies. The agitation did not influence the biosurfactant productivity, which reached its maximum value, using MRS medium as a nutrient source, before 48 h of incubation at a temperature of 35 °C. The glyco-lipo-peptide nature of the biosurfactant was confirmed by the analysis of its chemical composition. It showed stable surface activity in the pH range of 5-7 and a remarkable functionality as an emulsifier, especially in emulsions containing NaCl.

Prevalence, Antibio-Resistance and Risk Factors for Salmonella in Broiler Turkey Farms in the Province of Khémisset (Morocco)

Full-text available

Article

Jan 2014

The problem of contamination of poultry by salmonella in addition to the increase of antimicrobial resistance in Salmonella is of a great importance both in the field of public health as well as in the socioeconomic sector of the country because of the damage it can cause, but no studies have been conducted so far in Morocco to determine the risk factors of Salmonella contamination in broiler turkey farms. In order to determine the statute of the broiler turkey farms (n= 20) with respect to the contamination by Salmonella, three visits were paid to each livestock buildings. A batch of 10 pools of 5 droppings per breeding by visit were collected (n= 600) and analyzed. All Salmonella (n= 62) isolates were serotyped, confirmed by the presence of virulence gene (invasion) and tested for the resistance to 15 antimicrobial agents by the agar diffusion method. In parallel, an analytical study was carried out to investigate risks factors of Salmonella contamination in these farms. The rate of insulation of Salmonella is important (35 %) and the isolated serotypes are worrying: 36 S. Kentucky, 15 S. Saintpaul, 8 S. Parkroyal and 3 S. Ruzizi River. They have relatively high rates of resistance to tetracycline (79 %) and streptomycin (72.5 %), followed by resistance to nalidixic acid (37.1 %), ciprofloxacin (33.9 %), ampicillin (33.8 %), spectinomycin (32.3 %), trimethoprim (30.6 %) to trimethoprim-sulfamethoxazole (24.2 %), gentamicin (21 %), kanamycin (17.7 %) and amoxicillin + clavulanic acid (16.1%). Three strains of S. Agona expanded spectrum betalactamase producing which have a high level of resistance to ceftriaxone with a minimum inhibitory concentration (CMI) of 16 µg/ml. The variables associated with this contamination are related to the duration of crawlspace (p = 0,037), treatment with antibiotics (p = 0.001) and the contamination of turkeys poults (p = 0.002) dice implementation, The storage of manure inside the livestock building (p = 0.003), The conservation of turkeys inside the rearing building (p = 0,009) in the breeding season (p = 0.001) and age of turkeys sample (p = 0.01). The high level of antibiotic resistance of Salmonella isolates in the present study, showed the possible Significance of turkey as a source of multiple antimicrobial-resistant Salmonella for human infections.

Investigate the metabolites of Lactobacillus delbrueckii subsp. lactis isolated from local dairy (Yogurt) products in Iraq

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Experiment Findings

Jun 2022

s: Background and Objective: The production of different primary and secondary metabolites of probiotics,whose biological effects, metabolites of lactic acid bacteria offer therapeutic benefits in the prevention and treatment of various diseases, also responsible for the development of flavor, aroma and texture in food products in addition to their inhibitory activity for some pathogens such as fungi and pathogenic bacteria,The study aims to investigate the metabolites produced by Lactobacillus delbrueckii subsp.lactis as probiotics, its metabolites using Gc-Mass. Materials and Methods: Yogurt samples were collected from the local markets in Iraq for four local dairy products,The bacteria weresisolated from the dairy products using the Lactobacillus MRSsagar, Isolated species of dairy products were diagnosed based on phenotypic and microscopic characteristics,The isolate Lactobacillus delbrueckii subsp.lactis was chosen to study its metabolites using Gc-Mass. Results: Eight bacterial isolates were isolated and purified from local dairy products (L.delbrueckii subsp.lactis, Brevibacillus sp, B.paralicheniformis, B.adius ,Paenibacillus lautus ,B.licheniformis ,Bacillus spp and L.rhamnosus).The results of Gc-Mass analysis for supernatant of L.delbrueckii subsp.lactis showed that lactobacilli produce many diagnosed metabolic compounds,It produced thirty compounds including some organic acids, amino acids and alcohols. Conclusion: We have come to isolate and diagnose bacterial species(L.delbrueckii subsp.lactis, Brevibacillus sp, B.paralicheniformis, B.adius ,Paenibacillus lautus ,B.licheniformis .Bacillus spp and L.rhamnosus)from local dairy products in the local markets. The results of Gc-Mass analysis for supernatant of Lactobacillus delbrueckii subsp.lactis showed that lactobacilli produce many diagnosed metabolic compounds, it produced thirty compounds including some organic acids, amino acids and alcohols, which have an effective role in enhancing the flavor of food products.

Isolation and identification of keratinolytic probiotic Bucillus licheniformis bacteria from the soil below poultry slaughterhouse waste

Full-text available

Article

May 2022

Feathers make up 7% of the total weight of adult chickens and keratin protein makes up 85% of the feathers. Today, the keratinase enzymes of some Bacillus strains are used to degrade and process raw keratin waste for animal and poultry feed. According to various studies, the probiotic properties of some spore-shaped Bacillus have also been proven. The study aimed to isolation of the keratinolytic Bacillus bacteria that they have probiotic properties for using in the livestock and poultry feed industry. We were able to isolate 8 strains of Bacillus licheniformis with kreatin degrading properties from the soil of Baharan chicken slaughterhouse (Qom city, Iran) applying heat shock, alcohol- and keratin-rich culture medium, and after microscopic and biochemical analysis, 16S rDNA gene was isolated. The measurement results of keratinase activity showed that the three strains of Bacillus licheniformis pvkr6, pvkr 15, and pvkr41 had the highest activity with 124.08, 101.1, and 100.18 U/ml. The results of probiotic properties evaluation also revealed that among all the isolates, only Bacillus licheniformis pvkr15 and Bacillus licheniformis PTCC 1595 (positive control) were γ-hemolytic strains. The percentage of surface hydrophobicity of the strains was obtained from 3.27 to 30.57. It was also shown that, on average, all the strains had acceptable susceptibility to the tested antibiotics except penicillin G. Bacillus licheniformis pvkr15 with highest keratinase activity (101.1U/ml) was considered an optional probiotics due to its abilities such as (biofilm formation, being safe cause of γ-hemolytic activity, high susceptibility to antibiotics such as streptomycin, gentamicin, cefixime, amoxicillin, tetracycline, vancomycin, erythromycin and having a moderate hydrophilic (hydrophobicity: 19.09%), high survivability in pH 2, 2.5 and 3, strong resistance to bile salts and moderate antagonistic activity against pathogenic bacterium like Proteus mirabilis and the ability to grow under anaerobic conditions). By using this strain, after hydrolysis of keratin protein in the feather structure, to replace part of the protein of livestock and poultry feed, not only is no need to separate bacteria from the feed, but also the strain play role of an useful and effective additive in animal growth.

Exploring antimicrobial activity of Lactobacillus spp. (probiotics) isolated from raw cow's milk against Staphylococcus aureus causing bovine mastitis

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Article

Jan 2021

Bovine mastitis is a costly disease in the dairy farms globally. The control of such disease is generally based on the prevention by the strict hygienic measures during milking. Other approaches include vaccination and the application of antibiotics. Regardless to these procedures, mastitis is not entirely under the control, thus increasing the need for alternative tactics. This study was conducted to isolate and identify lactic acid bacteria (LAB) from fresh cow's milk which possess antibacterial activity that could be used for mastitis control. 146 isolates were recognized as (LAB) from105 milk tanks samples after being cultured anaerobically on de Man, Rogosa and Sharpe (MRS) agar plates for 48 hours at 37 o C and identified by general bacteriological investigation. Afterwards, 24 isolates were identified to belong to genus Lactobacillus using polymerase chain reaction (PCR), and for species level recognition MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) was used resulted in : L. fermentum (5), L. brevis (3), L. plantarum (4), L. paracasei (2), L. rhamnosus (3), L. pentosus (2), L. casei (3), L. raffinolactis (1) and L. mesenteroids (1). The antimicrobial activity of these strains against one of the major mastitis pathogens, S. aureus, was detected by the agar well diffusion assay and the modified double layer method , where L. casei, L. fermentum and L. plantarum possess the most inhibiting effect besides they have no hemolytic nor gelatin liquefaction activity when their safety profiles were evaluated. The result of the antibiotic susceptibility test revealed that these isolates were resistant to vancomycin (VA), neomycin (N) and gentamycin (CN). On the other hands, they were highly sensitive to amoxicillin clavulanic acid (AMC), Levofloxacin (LEV), tetracycline (TE) and penicillin (P). The study suggests that L. casei, L. fermentum and L. plantarum are perfect candidates to be used as probiotics to help in preventing and controlling bovine mastitis caused by Staphylococcus aureus as they were proven to be safe and have antimicrobial activity against the organism. ‫ـــــــــــــــــــــــــــــــــــــــــ‬ This is an open access article under the term of the Creative Commons Attribution 4.0 (CC-BY) International License. To view a copy of this license, visit: http://creativecommons.org/licenses/by/4.0/ J. Appl. Vet. Sci., 6 (3): 60-69.

Antimicrobial susceptibility pattern and the antagonistic effect of lactobacillus in fighting shigella spp. Isolated from diarrheic children

Full-text available

Article

Sep 2021

Background: Shigellais an major source of bacterial gastroenteritis in lack of health awareness in society. The handling of shigellosis mostly requires antibiotics. The using of probiotic of Lactic acid bacteria possess the counter effect against many dangerous bacterial pathogens which associated with gastroenteritis like shigella spp. Aim: the purpose of this study to estimate the influence of lactic bacteria of the genus Lactobacillus on the population Shigella as a main pathogen involved in gastroenteritis in children. Patients and methods: A total of 50 stool specimens were collected during the period September2019 to January 2020 from diarrheic children patients age range(1-3)years. Standard bacteriological methods were used to isolate, identify, and determine the antimicrobial susceptibility pattern of Shigellaisolates, and we used fresh culture of Lactobacillus 24 h (previously isolated as member of fecal microbiota from healthy person and identified by molecular assay). Then we done centrifugation to obtain supernatant which have test bioactive materials like bacteriocin. These bacteriocin materials subjected to own antibacterial activity against other bacterial pathogen like Shigella spp. By using agar diffusion method. Results: All the 14 Shigella spp. isolates show 100% resistance to nalidixic acid,. cotrimoxazole , and High resistance to ciprofloxacin (85%), and moderate resistant of ampicillin (64%). In agar-well diffusion method indicated the high antagonistic activity of the strains of Lactobacillus 2, 3, and 4 isolated from health GI tract against all Shigella spp., as a result of their activity the total elimination of Shigella within 24 h was observed. conclusion: the Shigella spp. Strains exhibited antibiotic resistant against more one type of antiobiotic The lactobacilii strains tested during this study showed strong antimicrobial activity against Shigella spp.

Influence of a Probiotic Yoghurt on the Status of Vitamins B1, B2 and B6 in the Healthy Adult Human

Full-text available

Article

Feb 2001

The main reason for this study was to determine whether yoghurt bacteria, being rich in some water-soluble vitamins, release them or utilize vitamins from their surroundings. Our study was trying to determine for the first time, if the viable bacteria of probiotic yoghurt are able to influence the parameters of the B-vitamin (B(1), B(2), B(6)) status of the healthy adult human. The test yoghurt was commercially available probiotic yoghurt prepared with Streptococcus thermophilus and Lactobacillus acidophilus, enriched with Lactobacillus casei GG. Different chemical forms of all investigated B-vitamins were determined by HPLC methods. In order to determine the influence of the yoghurt flora, each of 12 subjects consumed four yoghurt portions 125 g each ( = 500 g) a day, containing thermally inactivated cultures during the first 2-week period and yoghurt without heat treatment during the second 2-week period. The heat treatment of the probiotic yoghurt caused negligible changes in vitamin contents. The plasma levels of thiamin decreased significantly (p < 0.01) after the first 2-week period and kept on decreasing during the second 2-week period. A similar trend was found in the urinary excretion. The plasma levels of the B(2)-vitamers were different. The flavin adenine dinucleotide concentrations increased significantly (p < 0.01) after the consumption of heat-treated yoghurt and decreased significantly (p < 0.05) after the following 2 weeks, in which the subjects received the untreated yoghurt. In contrast, the flavin mononucleotide plasma levels decreased during the first 2-week period and increased during the second part of the study, but the change was not statistically significant. The free riboflavin concentrations in plasma and urine showed a continuous but not significant increase. The concentrations of pyridoxal-5-phosphate in plasma increased after the consumption of yoghurt with the inactivated bacteria and decreased in the second part of the study. However, the differences were not significant. The excretion of thiamin, B(2)- and B(6)-vitamers in the faeces did not significantly change throughout the study period (p>0.05). Our observations show that the bacterial flora of the examined yoghurt does not influence the vitamin B(1), B(2) and B(6) status of man. It seems likely that even lactobacilli of the 'probiotic' type which are vitamin B consumers can decrease the bioavailability of these vitamins for man. Obviously a thermal death of the cells did not induce a release of physiologically active vitamins.

Detection, purification, and partial characterization of plantaricin C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin

Full-text available

Article

Jul 1994

A bacteriocin produced by Lactobacillus plantarum LL441 was selected from the inhibitory products of 75 mesophilic lactobacilli because of its potency and broad spectrum. It is a peptide of 3.5 kDa whose amino-terminal sequence is NH2-K-K-T-K-K-N-X-S-G-D-I-. It is bactericidal and, in some cases, bacteriolytic. The peptide, called plantaricin C, retained its activity after boiling, storage, and treatment at different pHs.

Characterization of plantaricin KW30, a bacteriocin produced by Lactobacillus plantarum

Article

Dec 1996
J APPL MICROBIOL

A protease-sensitive antibacterial substance, produced by a strain of Lactobacillus plantarum isolated from fermented corn, was classified as a bacteriocin and designated plantaricin KW30. The bacteriocin was stable to heat, pH and treatment with surfactants, and unaffected by -amylase, lipase or lysozyme. Plantaricin KW30 exhibited a bactericidal and non-bacteriolytic mode of action against indicator cells, and inhibitory activity was limited to other lactobacilli. Maximum production was in MRS broth, and coincided with the onset of stationary phase under conditions of low pH and high cell numbers. In a complex medium bacteriocin production was enhanced by the presence of sodium acetate and Tween 80. Curing experiments gave derivatives that no longer produced the bacteriocin but retained immunity to it. These Bac derivatives showed the same plasmid pattern as the parent strain suggesting a chromosomal location for the genes for bacteriocin production.

Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome C oxidase containing proteoliposomes

Article

Sep 1991

The interaction of the peptide antibiotic nisin with liposomes has been studied. The effect of this interaction was analyzed on the membrane potential (inside negative) and the pH gradient (inside alkaline) in liposomes made from Escherichia coli phosphatidylethanolamine and egg phosphatidylcholine (9:1, wt/wt). The membrane potential and pH gradient were generated by artificial ion gradients or by the oxidation of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, and cytochrome c by the beef heart cytochrome c oxidase incorporated in the liposomal membranes. Nisin dissipated the membrane potential and the pH gradient in both types of liposomes and inhibited oxygen consumption by cytochrome c oxidase in proteoliposomes. The dissipation of the proton motive force in proteoliposomes was only to a minor extent due to a decrease of the oxidase activity by nisin. The results in these model systems show that a membrane potential and/or a pH gradient across the membrane enhances the activity of nisin. Nisin incorporates into the membrane and makes the membrane permeable for ions. As a result, both the membrane potential and pH gradient are dissipated. The activity of nisin was found to be influenced by the phospholipid composition of the liposomal membrane.

Probiotics, prebiotics, and synbiotics: Approaches for modulating the microbial ecology of the gut

Article

Jun 1999

The microbiota of the human large intestine influences health and well-being. Whereas it has long been accepted that gut bacteria play a role in host pathogenesis, current opinion is that certain microflora components can have beneficial effects on gastroenteritis resistance, blood lipids, antitumor properties, lactose tolerance, and gastrointestinal immunity. It is postulated that in the infant gut an elevated bifidobacterial count may be associated with health advantages that breast-fed infants may have over formula-fed infants. Whereas beneficial aspects of the human gut flora still need definitive confirmation and mechanistic explanations, there is now interest in modulating the composition of gut flora such that a potentially more remedial community exists. This may be achieved through the targeted use of dietary supplementation. This article provides an overview of how probiotics, prebiotics, and synbiotics may contribute toward nutritional modulation of the gut microecology, with emphasis on the neonatal intestine where appropriate. The use of modern molecular methods, as an essential step forward for assessing the validity and accuracy of the modulatory approach, is also discussed.

Probiotic Bacteria: Today and Tomorrow

Article

Mar 2000

Todd R. Klaenhammer

This paper provides an overview of the key issues raised during this symposium. Probiotic cultures have been associated historically with cultured milks and dairy products, from which there is substantial evidence for positive effects on human health and general well-being.

Prebiotics and probiotics: Are they functional foods?

Article

Jul 2000

M Roberfroid

A probiotic is a viable microbial dietary supplement that beneficially affects the host through its effects in the intestinal tract. Probiotics are widely used to prepare fermented dairy products such as yogurt or freeze-dried cultures. In the future, they may also be found in fermented vegetables and meats. Several health-related effects associated with the intake of probiotics, including alleviation of lactose intolerance and immune enhancement, have been reported in human studies. Some evidence suggests a role for probiotics in reducing the risk of rotavirus-induced diarrhea and colon cancer. Prebiotics are nondigestible food ingredients that benefit the host by selectively stimulating the growth or activity of one or a limited number of bacteria in the colon. Work with prebiotics has been limited, and only studies involving the inulin-type fructans have generated sufficient data for thorough evaluation regarding their possible use as functional food ingredients. At present, claims about reduction of disease risk are only tentative and further research is needed. Among the claims are constipation relief, suppression of diarrhea, and reduction of the risks of osteoporosis, atherosclerotic cardiovascular disease associated with dyslipidemia and insulin resistance, obesity, and possibly type 2 diabetes. The combination of probiotics and prebiotics in a synbiotic has not been studied. This combination might improve the survival of the bacteria crossing the upper part of the gastrointestinal tract, thereby enhancing their effects in the large bowel. In addition, their effects might be additive or even synergistic.

Partial characterization of a bacteriocin produced by Lactobacillus delbrueckii subsp lactis UO004, an intestinal isolate with probiotic potential

Article

Sep 2001

The partial characterization of a bacteriocin produced by a human Lactobacillus delbrueckii isolate with probiotic potential. A bacterocin, UO004, was partially purified by cation exchange followed by a hydrophobic interaction column, biochemically characterized and the N-terminal region sequenced. Bacteriocin UO004 was found to be a hydrophobic, heat-stable polypeptide with an apparent molecular mass of 6 kDa. It was also stable and active over a wide pH range. The active compound was proteinaceous, heat-stable, and had a bactericidal (and bacteriolytic) mode of action on a limited number of micro-organisms. Such a narrow spectrum of activity is typical for bacteriocins produced by intestinal Lactobacillus. Bacteriocin UO004 from a probiotic strain is a new compound that does not share any homology with any other known lactic acid bacteria bacteriocin. Furthermore, Lact. delbrueckii is regarded as a suitable starter for the production of fermented milks.

Antagonistic activity of Lactobacillus bacteria strains against anaerobic gastrointestinal tract pathogens (Helicobacter pylori, Campylobacter coli, Campylobacter jejuni, Clostridium difficile)

Article

Feb 2001

Antagonistic activity of Lactobacillus strains has been known for some time. This property is connected with production of many active substances by lactobacilli e.g., organic acids and bacteriocin-like substances which interfere with other indigenous microorganisms inhabiting the same ecological niche, including also anaerobic gastrointestinal tract pathogens. Growing interest of clinical medicine in finding new approaches to treatment and prevention of common inflammatory infections of the digestive tract resulted in studies on a possible usage of lactic acid bacteria. Last years, several in vitro and in vivo experiments on antagonism of different Lactobacillus strains against Helicobacter pylori and Clostridium difficile were performed. These observations had been done on already established, well known probiotic Lactobacillus strains. We tested antibacterial activities of Lactobacillus strains isolated from human digestive tract. As indicator bacteria, four species known as anaerobic bacterial etiologic agents of gastroenteric infections: Helicobacter pylori, Campylobacter jejuni, C. coli and Clostridium difficile were used. Some of them were obtained from international collections, others were clinical isolates from specimens taken from patients with different defined gastrointestinal infections. We used a slab method of testing inhibitory activity described in details previously. Following conclusions were drawn from our study: All tested human Lactobacillus strains were able to inhibit the growth of all strains of anaerobic human gastrointestinal pathogens used in this study. Inhibitory activities of tested Lactobacillus strains against Helicobacter pylori, Campylobacter spp., and Clostridium difficile as measured by comparing mean diameters of the inhibition zones were similar. Differences in susceptibility of individual indicator strains of Campylobacter spp. and Clostridium difficile to inhibitory activity of Lactobacillus strains were small. A similar mechanism of inhibition of anaerobic bacteria by lactobacilli is postulated.

Probiotics - Possibilities and limitations of their application in food, animal feed, and in pharmaceutical preparations for man and animals

Article

Nov 2001
BERL MUNCH TIERARZTL

G Reuter

Probiotics are cultures of special microorganisms, which have been used as feed additives since the seventies of the past century and already since the twenties in food specimen and in pharmaceuticals. The definition of "Probioticum" was formulated in 1974 simultaneously with the use of living cultures in feed for various animals in order to substitute the application of nutritive antibiotics or chemotherapeutics. In the meantime probiotics are applied not only as feed supplements or pharmaceuticals but increasingly in suitable food specimens such as dairy products, fruit juices, chocolates, and even meat products. Of course, heating of such products prior to consumption or application must be ruled out. The selection of a suitable strain of a microorganism can be regarded as the primary requirement for the use as a probiotic. These cultures must be able to pass the stomach-duodenum barrier in a viable state and to multiply at the site of destination in the intestine. Additionally, they must be capable of producing antagonistic metabolites against a dominating saprophytic microflora resulting in a competitive growth. These abilities are common among lactic acid bacteria, e.g. lactobacilli and bifidobacteria. Yet, intended autochthonous species do not possess very good technological features for the enrichment in food specimens, because their viability will decrease rapidly under unfavourable conditions as it is the case with mineral supplements in feed or with low acidity in fermented dairy products. Therefore, some other microorganisms like spore-forming species or yeast cultures were introduced as probiotic components. These possess some similar features which render them suitable for probiotic use. But their physiological and ecological traits do not qualify them as probiotics of first choice. The special efficacy of probiotics must be strictly verified in animal nutrition due to restrictive EC-regulations, in pharmacy due to legal restraints, and in food applications in accordance with food law regulations. Safety aspects are considered very restrictively in feed applications, in the food and pharmaceutical sector they should be in accordance with the intended purpose of "fulfilling health claims". In the presented review the different requirements for the application of probiotics in animal nutrition, in food, and in pharmaceuticals will be provided. The special effect of competitive exclusion of pathogenetic and toxinogenic microorganisms in fowl performance are mentioned and even the application of probiotics as marine aquacultures of fish and Crustaceae will be included. Furthermore, the safety aspects resulting from the tremendous amount of industrially produced cultures which are distributed into the environment will be discussed. In conclusion it may be pointed out that probiotics may serve to partially replace the presently reduced or even prohibited application of nutritive antibiotics or chemotherapeutics in animal nutrition and in fulfillment of health claims in man and animals. Economic and environmental aspects will reduce the overall application of probiotics world-wide. Restrictions of use and controls of efficacy and safety are essential and must be implemented periodically.