assessment of potential probiotic properties of lactic acid bacteria and ...

FOOD BIOTECHNOLOGY Vol. 16, No. 3, pp. 211–225, 2002

ASSESSMENT OF POTENTIAL PROBIOTIC PROPERTIES OF LACTIC ACID BACTERIA AND YEAST STRAINS Velitchka Gotcheva,1 Eli Hristozova,3 Tsonka Hristozova,4 Mingruo Guo,2 Zlatka Roshkova,1 and Angel Angelov1,* 1

Higher Institute of Food and Flavor Industries, Plovdiv, Bulgaria 2 University of Vermont, Burlington 05405, USA 3 Medical Academy, Plovdiv 4 Institute of Microbiology, Plovdiv

ABSTRACT Four lactic acid bacteria (LAB) and three yeast strains isolated from a traditional Bulgarian cereal-based fermented beverage were assessed for potential probiotic properties. Acid and bile resistance, antipathogenic activity and antibiotic resistance of the strains were evaluated. Tolerance to low pH values (2.0 3.0) and high bile concentrations (0.2 2.0%) of the LAB and yeast strains varied, but all strains kept viable throughout the experiments. Antagonistic activity towards most of the eight testpathogens was observed for one LAB (Lactobacillus plantarum B28) and two yeast strains (Candida rugosa Y28 and Candida lambica Y30). Antibiotic resistance (39 antibiotics) of the LAB strains was variable, but showed their potential for therapeutic application. Key Words:

Lactic acid bacteria; Yeast; Probiotic properties

*Corresponding author. Fax: 359 32 651 498; E-mail: [email protected] 211 DOI: 10.1081=FBT-120016668 Copyright # 2002 by Marcel Dekker, Inc.

0890-5436 (Print); 1532-4249 (Online) www.dekker.com

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INTRODUCTION Functional foods remain the hottest topic in the food industry and nutrition, especially food products containing probiotic microorganisms.[1] Lactic acid bacteria (LAB) strains of genus Lactobacillus have been used for a long time for the production of a wide range fermented dairy and cerealbased products and have been given generally-recognized-as-safe (GRAS) status. Probiotics are commonly de®ned as viable microorganisms that, upon ingestion in certain numbers, exert health bene®ts beyond inherent basic nutrition.[2] Probiotics are a desirable natural tool to maintain the healthy balance of the human intestinal micro¯ora. They are consumed either as food components or as non-food preparations. New species and more speci®c strains of probiotic bacteria are constantly identi®ed. However, prior to incorporating strains into products, their ecacy should be carefully assessed. To prove scienti®cally the probiotic properties of these strains, they have to ful®l series of selective criteria. First, the probiotic strains have to be safe for human consumption. Research has focused on natural isolates with a long history of safe use (e.g., they originate either from traditionally consumed foods with proven health eects or from the gastro-intestinal system of healthy individuals). Acid and bile stability are self-evident properties for every strain expected to have eects in the intestinal tract. Development of ecacious probiotic products suggests as well that the strains should have characteristic antimicrobial activity and antibiotic resistance. Adhesion and colonization of intestinal cells are desired properties, closely related to potential immune eects of the strains studied.[3 6] Acidic conditions in the stomach are a natural barrier preventing most microorganisms from passing into the intestines. Normally, pH in the stomach of healthy individuals is lower than 3.0, while within an achloric stomach (for people with anemia) pH is over 6.0. A few bacteria can tolerate environment of pH lower than 3.0. Prior to feeding, the amount of bacteria in the stomach is up to 103 colony-forming units (CFU)=mL gastric juice, while after food intake, their counts increase to 105 CFU=mL. The ability to survive and grow in low pH environment is characteristic for LAB and yeasts.[7] The tolerance mechanism of LAB and yeasts to these conditions is not clari®ed completely yet. Production of organic acids by LAB decreases pH of the environment, which becomes selective against the microorganisms sensitive to low pH. Bile tolerance of microorganisms has been used as a selective criterion for potential probiotics.[8] Some reports show strong eect of bile salts on the survivability of dierent strains, but this depends on the bile concentrations as well as the speci®c properties of the strains.[7] The stress eect of bile on Lactobacillus strains is complex because of the variation of bile concentration and passage time within the dierent parts of

PROBIOTIC PROPERTIES OF LAB AND YEAST STRAINS

213

the gastro-intestinal tract.[9] It has been estimated that some strains survive higher than normal bile salts concentrations.[8] However, similar studies on yeasts have not been reported yet. Bile resistance of some strains is related to speci®c enzyme activity bile salt hydrolase (BSH) which helps hydrolyze conjugated bile, thus reducing its toxic eect.[10] In addition, according to Ganzle et al.,[11] bile resistance can be increased due to the protective eect of some food components. An important property of the probiotic strains is their antagonistic activity against pathogenic bacteria either by competitive exclusion, decrease of redox potential, interbacterial aggregation, or production of antimicrobial substances including organic acids, other inhibitory primary metabolites such as hydrogen peroxide, and special compounds like bacteriocins and antibiotics.[1,12] This property plays a signi®cant role in the ability of probiotics to compete against the resident intestinal ¯ora and bene®cially modify it. Numerous clinical studies demonstrate that organisms of the Lactobacillus genus are both preventative and therapeutic in controlling intestinal infections when administered with milk containing these organisms.[13] Antibiotic resistance of probiotic strains assures maintenance of healthy intestinal microbiota throughout antibiotic treatments of microbial infections. LAB display a wide range of antibiotic susceptibilities and resistances. Isolates of lactobacilli with strong resistance to penicillins, cephalosporins and bacitracin have been recovered from human gastro-intestinal tract samples and dairy products. In most cases, antibiotic resistance is not transmissible, but represents an intrinsic characteristic of the organism. An important requirement for probiotic strains is that they do not harbor mobile elements carrying resistance genes. Some authors report that many intrinsically vancomycin-resistant strains of lactobacilli have been safely used as probiotics with no indication of transferring this resistance to other species.[4,14] The objectives of this study were to characterize lactic acid bacteria and yeast strains, isolated in previous research from a traditional Bulgarian cereal-based fermented beverage according to the requirements for probiotics in order to consider their further application in the development of new functional products. MATERIALS AND METHODS Strains and Culture Conditions Several LAB and yeast strains isolated in previous studies[15] were tested: strain Lb. plantarum ssp. pseudoplantarum (B3), two strains of Lb. plantarum (B25 and B28), one of Lb. paracasei ssp. casei (B29), Trichosporon cutaneum (Y27), Candida rugosa (Y28), and Candida lambica (Y30). LAB strains were maintained in MRS broth (Difco, Difco Laboratories, Detroit, MI) at 37 C for 48 h. Yeast strains were grown in ME broth (Oxoid,

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Hampshire, UK) at 30 C for 24 h. For long-term storage, strains were kept at 40 C in 15% glycerol. All strains were subcultured twice prior to experiments. Acid Resistance MRS and ME broths at pH 2.0, 2.5 and 3.0 were prepared and inoculated with 1% (v=v) 0.5 McFarland overnight suspensions of the tested LAB and yeast strains. Control samples without acidi®cation were also prepared. Incubation was carried out at 37 C and 30 C, respectively, and absorbance of the samples was measured every 30 min at 560 nm against the corresponding non-inoculated blanks according to Rossi et al.[16] In both tests, plate counts (MRS agar and ME agar) from the initial and the ®nal suspensions were made to assess survivability when growth was not clearly indicated by change of absorbance at the certain conditions. Bile Resistance MRS-broth (Difco) and ME broth (Oxoid) supplemented with 0.2%, 0.3%, 0.4%, and 2.0% (w=v) oxgall (dehydrated fresh bile) (Difco) were prepared and inoculated with 1% (v=v) 0.5 McFarland overnight suspensions of the tested LAB and yeast strains, respectively. Control samples without oxgall were prepared as well. LAB and yeasts samples were incubated at 37 C and 30 C, respectively. Absorbance was measured (DU-64 Spectrophotometer, Beckman, Chaska, MN) every 30 min at 560 nm against the corresponding non-inoculated blanks. Antimicrobial Activity The agar spot test method was performed for testing potential antimicrobial activity of LAB strains according to the method of Barry and Thornsberry[17] with eight strains of clinical pathogen isolates from patients Salmonella enteritidis 1927, Salmonella enteritidis 1968, Pseudomonas aeruginosa 1806, Pseudomonas aeruginosa 1831, E. coli O27, E. coli O26, E. coli O6, and Enterococcus. The test was performed on Bacto Nutrient Agar (Difco).[18] The presence=absence of inhibition zones was observed after 24 h of incubation of the plates at 37 C. A clear zone of more than 1 mm around a spot was accepted positive. Resistance to Antibiotics The agar plate dilution method was applied. Muller-Hinton agar (Oxoid) was used as the basal medium for bacterial growth. Culture suspensions were


215

obtained after incubation at 37 C in MRS broth and spread on the agar plates at 0.5 McFarland. Standard discs (BBL, SmithKline Beecham, UK) with thirty-nine antibiotic substances were applied by multipositional disc dispenser. The plates were incubated 24 h at 37 C. The inhibition zones were read according to the Bauer-Kirby's 3-level system[19] and were compared with the values of NCCLS.[20] Validation of the test was done by control antibiograms with reference strains E. coli ATCC 25 922, S. aureus ATCC 25 923, Ps. aeruginosa ATCC 27 853. Data Analysis Each experiment was performed in triplicate. Data were submitted to one-way ANOVA with a least signi®cant dierence of 95%. RESULTS AND DISCUSSION Acid Resistance Acid tolerance is a property that any strain expected to have eects in the gastrointestinal tract should possess.[3,7] Resisting at pH 3.0 for 2 h is one standard for low pH tolerance of probiotic bacteria.[21] In this study, behavior of the four LAB and three yeast strains at low pH 2.0 3.0 varied. The initial absorbance (A) of LAB samples was within 0.04 0.09. For 4.5 h, growth of all strains at pH 2 was strongly inhibited (Table 1). The agar plating, however, showed that the strains remained viable for this period. Signi®cant changes of absorbance (P < 0.05) were observed for strains B25, B28 and B29 at pH 2.5 and 3.0. Absorbance of B3 samples at pH 2, 2.5 and 3.0 did not change signi®cantly and only agar plates showed survival at the end of the experiment. Strain B28 showed best resistance, followed by B25. Jacobsen et al.[22] observed survival of 29 out of 44 LAB strains (Lactobacillus spp.), following 4 h of incubation at pH 2.5, but replication of the results was questionable. Yeast strains showed much lower tolerance to the test conditions (Table 1), as expected. After 4.5 h at repressing conditions, changes of absorbance were not signi®cant and the parameter observed did not indicate growth of any of the strains tested. However, agar plating at the end of the experiment showed survival of the strains. The results indicated that the strains could survive in the acidic stomach environment and reach the areas of bene®cial activity (small intestine and colon) when ingested. Acid tolerance of the strains could be improved by some natural protectors within the product consumed, such as protein in dairy products.

0,245 0,003 0,028 0,013

Control pH 2.0 pH 2.5 pH 3.0

S

0,265 0,008 0,050 0,014

DA560

B25

S 1,556 0,001 0,058 0,022

DA560

B28

S 1,109 0,01 0,018 0,022

DA560

B29

S

Y27

0,008 0,039 0,033 0,013

DA560

Assessment of Acid Tolerance

S

0,036 0,006 0,001 0,004

DA560

Y28

S

0,067 0,026 0,019 0,015

DA560

Y30

S

Results represent change of absorbance (DA560) within 270 min, based on the means of three independent measurements, and survival (S) of the strains tested by agar-plating after the test time.

DA560

Strains

B3

Table 1.

216 GOTCHEVA ET AL.


217

Bile Resistance After passage through the acidic stomach conditions, it is important that, for application of LAB strains as probiotics, they are able to survive the bile salt in the intestine, the normal level of which is around 0.3%, but may range up to the extreme 2.0% during the ®rst hour of digestion. Most researchers assess bile resistance within the range of 0.1 0.5%. Some, though, used bile concentrations ranging from 0.5 to 2.0% for the selective isolation of bile tolerant bacteria from mixed cultures.[23] Charteris et al.[24] reported the good bile tolerance (0.3% v=v) of 7 Lactobacillus and 7 Bi®dobacterium strains in an in vitro study. Jacobsen et al.[22] studied the survival of LAB strains tested for 4 h in 0.3% oxgall. Several Lactobacillus and Bi®dobacterium strains were tested at bile levels of 1.0 and 1.5%. Some strains kept counts unaected after 3 h of incubation at these bile levels.[23] LAB strains studied hereby showed tolerance to bile salts due to their BSH activity (Fig. 1). Changes of absorbance for strains B3 and B25 were signi®cant (P < 0.05) at 0.2 and 0.3% bile concentrations. Better results were observed for strains B28 and B29, where absorbance did not change

Figure 1.

Assessment of bile tolerance±LAB strains.

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GOTCHEVA ET AL.

Figure 2.

Assessment of bile tolerance±yeast strains.

signi®cantly at the extreme 2.0% bile only. Strain B3 was least tolerant. However, agar plating indicated survival of all LAB strains at the end of the test time. Changes of absorbance for Y27 were signi®cant (P < 0.05) at 0.2 and 0.3% bile, while for Y28 and Y30 absorbance at 0.2% were only signi®cant. Y27 showed better tolerance, followed by Y30 and Y28. However, yeast strains remained viable at all bile concentrations throughout the test (Fig. 2), indicated by agar plates. This indicates they could survive bile toxicity during their passage through the gastro-intestinal system. Results from bile resistance evaluation of the LAB and yeast strains increased their potential for eventual applicability in functional foods development. As strain B3 showed lowest acid and bile tolerance, it was excluded from the further tests. Antimicrobial Activity The potential control of intestinal pathogens by lactic acid bacteria and yeast strains with probiotic properties is a valuable feature for considering


219

their application in functional food development. Some strains have been shown to be both preventive and therapeutic in controlling intestinal infections. A study of Koga et al.[25] showed no antimicrobial activity of the LAB tested towards pathogenic strains Salmonella enteritidis and E. coli. Jacobsen et al.[22] studied LAB isolates from fermented maize, strains with documented antimicrobial properties, human clinical isolates and dairy strains. Their activities varied too, many of the strains showing weak or no inhibition of the test-pathogens. In the present study, the inhibitory activity of the LAB and yeast strains towards pathogens was tested with eight clinical isolates: two obligatory pathogens S. enteritidis 1927 and 1968, two strains, causing opportunistic infections Ps. aeruginosa 1806 and 1831, one conditionally pathogenic Enterococcus, and three strains E. coli the entheropathogenic E. coli O26 and entherotoxigenic E. coli O6 and O27. The antimicrobial activities of the LAB strains tested were variable. Strain B25 had some activity towards Salmonella. Strain B29 showed no inhibition, while B28 had a clearly de®ned activity towards 6 of the testpathogens, especially Salmonella. Of the yeast strains, Y27 had no antagonistic activity, while Y28 showed good inhibition of the Salmonella strains and Ps. aeruginosa 2. Y28 was active towards E. coli O6 and O26 as well. Strain Y30 behaved in a similar way, actively inhibiting Salmonella and Ps. aeruginosa and eective to E. coli O26 and O27. Results indicate the good potential of strains B28, Y28 and Y30 to deliver an antipathogen barrier when applied in a food product such as yogurt or fermented cereals. Antibiotic Resistance Application of antibiotics often disrupts the healthy balance of the residential micro¯ora of the host, causing intestinal disorders. The administration of antibiotic-resistant strains can help keeping the normal bacterial ratio in the intestines, or its fast restoration if applied after the antibiotic treatment. Resistance of the three LAB strains to 39 antibiotics and chemotherapeutics was tested. In vitro resistance (R) and intermediate sensitivity (I), which, at in vivo conditions, turns into resistance, were observed towards 21 of the test substances. All three strains were resistant to the inhibitors of the cell wall synthesis penicillinase resistant Oxacillin (1 mg) and Methicillin (5 mg), as well as to the cephalosporin generation II Cefotoxine (30 mg) and the glycopeptides Vancomycin (30 mg) and Teicoplanine (30 mg). Vancomycin resistance is an intrinsic property of many LAB,[14] suggesting resistance of the tested LAB towards this antibiotic.

0 0.0 2 0.3 0 0.0 0 0.0 1 0.0 0 0.0 0 0.0 0 0.0

B25 Growth Zone (mm) 10 7 1 6 5 4 4 1

0.5 0.5 0.0 0.0 0.0 0.5 0.0 0.0

B28 Growth Zone (mm) 0 0 0 0 0 0 0 0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

B29 Growth Zone (mm) 0 0 0 0 0 0 0 0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Y27 Growth Zone (mm)

Test Strain

7 8 0 7 2 2 0 0

0.5 0.0 0.0 0.5 0.5 0.5 0.0 0.0


Antagonistic Activities of Bacterial and Yeast Strains Against Selected Bacterial Pathogens

6 1.0 8 1.0 0 0.0 5 0.0 0 0.0 4 0.0 2 0.0 0 0.0


Results represent the means of three independent measurements with standard deviation. A clear zone of more than 1 mm around an inoculated agar well was accepted positive.

Salmonella enteriditas 1 Salmonella enteriditas 2 Pseudomonas aeruginosa 1 Pseudomonas aeruginosa 2 E. coli O6 E. coli O26 E. coli O27 Enterococcus

Pathogen

Table 2.

220 GOTCHEVA ET AL.

Penicillin G Oxacillin Ampicillin Amoxicillin Cloxacillin Azlocillin Piperacillin Carbenicillin Amoxicillin=Clavulonic acid Ampicillin=Sulbactam Cephalexin Cefoxitine Cefuroxime Cefazolin Ceftazidime Ceftriaxone Cefepime Imipenem Meropenem Amikacin Gentamicin Tobramycin Kanamycin Tetracyclin Doxycyclin

Antibiotic 10 1 10 10 1 75 100 100 30 20 30 30 30 30 30 30 30 10 10 30 10 10 30 30 30

Interpretation S R S S R S ND ND S S R R S S R S S S S R S R R S S

Zone (mm) 36 1 60 30 1 40 0 60 20 1 ND ND 36 1 24 1 60 60 28 1 38 1 80 32 1 18 0 28 1 24 1 10 0 16 1 60 60 34 1 36 1

Strain B25

15 1 60 18 1 60 60 20 1 38 1 28 1 22 1 15 0 22 1 60 20 0 30 1 19 1 20 1 30 1 40 1 30 1 60 60 60 60 20 1 20 1

Zone (mm) R R R R R S S S S S S R S S S I S S S R R R R S S

Interpretation

Strain B28

Antibiotic Resistance Patterns of Selected Lactobacillus Strains

Disc Potency (mg)

Table 3.

20 1 60 ND 60 60 40 1 44 1 40 1 30 1 20 1 ND 60 24 1 36 1 18 0 20 1 24 1 44 1 32 1 12 1 12 1 60 60 22 1 38 1

Zone (mm)

(continued )

I R ND R R S S S S S ND R S S S I S S S R R R R S S

Interpretation

Strain B29

PROBIOTIC PROPERTIES OF LAB AND YEAST STRAINS 221

38 40 20 36 34 30 30 6 20 16 22 6 6 40

5 5 30 5 5 5 30 30

S S S S S S S R S I S R R S

1 0 0 1 0 0 0 1

Interpretation

1 1 1 1 1 1

Zone (mm)

30 15 15 2 2 1.25=23.7

Disc Potency (mg)

14 1 60 60 60 60 60 10 1 30 1

20 1 26 1 22 1 30 1 36 1 60

Zone (mm)

R R R R R R R S

S S S S S R

Interpretation

Strain B28

24 1 60 10 1 60 12 1 60 60 40 1

36 1 30 1 20 1 32 1 34 1 60

Zone (mm)

R R R R R R R S

S S S S S R

Interpretation

Strain B29

S ¼ susceptible; I ¼ intermediate; R ¼ resistant; ND ¼ not determined. Results represent the means of three independent measurements with standard deviation. S, I and R were interpreted accordingly for each individual test-antibiotic from BBL interpretation tables, following the NCCLS [20]. When the three values were incompatible, reaction towards the antibiotic was not determined (ND).

Chloramphenicol Erythromycin Clarythromycin Clindamycin Lincomycin TrimethoprimSulfamethoxazole Rifampin Nalidix acid Ciprofloxacin Pefloxacin Ofloxacin Vancomycin Teicoplanin Piperacillin=Tazobactam

Antibiotic

Strain B25

Table 3. Continued

222 GOTCHEVA ET AL.


223

Strain B25 had a limited resistance to cephalosporins generation III and a well de®ned one to the inhibitors of protein synthesis, aminoglycosides Amikacin (30 mg), Tobramycin (10 mg) and Kanamycin (30 mg). Strain B28 showed full resistance to all inhibitors of nucleic acid synthesis Nalidix acid and chynolons, as well as to Trimethoprim-Sulfamethoxazole 1.25=23.75 mg, which indicates good probiotic potential. Strain B29 was only partially resistant to inhibitors of the cell wall synthesis and showed good resistance to aminoglycosides, quinolones and Trimethoprim-Sulfamethoxazole. Antibiotic resistances of the strains indicate their potential to be applied in therapeutic treatments.

CONCLUSIONS The LAB and yeast strains evaluated in the study have a long history of safe human consumption. The study on their potential probiotic properties showed that survivability of the strains in the conditions of high oxgall concentration (up to 2.0%) and low pH (2.0) varied, but they still remained viable throughout the tests. This suggests that they could reach the small intestine and the colon, thus contributing the balance of the intestinal micro¯ora. The antimicrobial activity of strains B28 and Y28 towards the pathogen strains used in the study can be bene®cial as providing preventative or=and therapeutic eect when applied in a food product and ingested. The resistance of the LAB strains to some antibiotics is an indicator for their potential to minimize the negative eects of antibiotic therapy on the host bacterial ecosystem. The strains studied seem to have good potential for application in functional foods and health-related products. More research is needed on other potential probiotic properties of the strains such as ability to adhere and colonize human intestinal cells.

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