Activity and viability of probiotic candidates consisting of lactic acid bacteria and yeast isolated from native poultry gastrointestinal tract Rima Shidqiyya Hidayati Martin, Erika Budiarti Laconi, Anuraga Jayanegara, Ahmad Sofyan, and Lusty Istiqomah
Citation: AIP Conference Proceedings 2021, 070012 (2018); doi: 10.1063/1.5062810 View online: https://doi.org/10.1063/1.5062810 View Table of Contents: http://aip.scitation.org/toc/apc/2021/1 Published by the American Institute of Physics
Activity and Viability of Probiotic Candidates Consisting of Lactic Acid Bacteria and Yeast Isolated from Native Poultry Gastrointestinal Tract Rima Shidqiyya Hidayati Martin1, Erika Budiarti Laconi1,a), Anuraga Jayanegara1, Ahmad Sofyan2,b) and Lusty Istiqomah2 1
Nutrition and Feed Technology Department, Faculty of Animal Science, Bogor Agricultural University,, Bogor 16680, West Java, Indonesia 2 Laboratory of Bio-Feed Additive Technology, Research Unit for Natural Product Technology, Indonesian Institute of Sciences, Yogyakarta 55861, Indonesia. a)
Corresponding author:
[email protected] b)
[email protected]
Abstract. Live microbial cultures have a positive effect on animal digestibility by increasing the natural balance of microflora in the digestive tract. This study evaluated lactic acid bacteria (LAB) and yeast as probiotic candidates with resistance to bile salt, acid conditions, and inhibitory activities against pathogenic bacteria. The LAB and yeast were isolated from the colons of native chicken and Javanese duck, respectively. This experiment followed a factorial design. Characteristics of the isolates were evaluated as follows: antibacterial activity, antibiotic sensitivity, and viability percentage in bile salt, gastric juice and acid conditions. The result showed that both isolates had the ability to inhibit Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella pullorum. The yeast B-18 isolate was resistant to streptomycin 10 μg, penicillin 10 μg, and erythromycin 15 μg, while the LAB AKK-30 isolate was resistant to streptomycin 10 μg and penicillin 10 μg. LAB AKK-30 tends to be tolerant to bile salt, while yeast B-18 was more resistant to gastric juices and acid conditions. It concluded that both isolates are potentially useful probiotic candidates followed by microencapsulation. Further experiments should be conducted to evaluate the effectiveness of the isolates on nutrient use in poultry. Keyword: Isolate, lactic acid bacteria, microbial cultures, resistant
INTRODUCTION The use of antibiotics as a growth promoter (AGP) has a positive impact on animal performance by decreasing the population of sensitive bacteria in the gastrointestinal tract (GIT) so animals digests more efficiently.1 AGP can reduce GIT infections and increase muscle mass.2 On the other hand, AGP has the negative effect of increasing the resistance of gastrointestinal bacteria and leaving residues on animal products.3,4 Long-term use of AGP causes bacteria in the GIT to become resistant to antibiotics, preventing treatment of infections. Humans become resistant if they consume animal products contaminated by the bacteria containing the resistant gene and residue form AGP on animal products results in allergic or toxic reactions in the human body. As a result, AGP has been banned since 2006; probiotics are the alternative solution. Probiotics can be isolated from the GIT of poultry that has abundant and dominant microbiota. Microbiota from the crop, gizzard, duodenum, jejunum, ileum, caecum, and feces/excreta have been studied while microorganisms from the colon have seldom been studied. The colon has been considered the part of the GIT with less varied microbiota, whereas this section potentially contains probiotic microorganisms. Native poultry, for example chickens and ducks from Indonesia, have been known for their ability to survive on pathogenic bacteria and for their high adaptation to the environment. This might be a result of microbiota in their The 8th Annual Basic Science International Conference AIP Conf. Proc. 2021, 070012-1–070012-6; https://doi.org/10.1063/1.5062810 Published by AIP Publishing. 978-0-7354-1739-7/$30.00
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GIT. Hence, this study uses microorganisms isolated and identified from the colons of native poultry. The isolate from native chicken colon was lactic acid bacteria, while the isolate from Javanese duck colon was yeast. Both isolates are examined as probiotic candidates. Examination of probiotic candidates is adjusted to the characteristics of the probiotic. These characteristics are related to the probiotic ability to survive in the GIT. Most probiotics have the ability to control and prevent pathogenic bacteria colonization, so probiotics become dominant in the GIT.5 The GIT contains mucus produced by gastrointestinal organ, which is neutral, and acids such as bile salt and gastric juices. The mucus becomes a barrier for some bacteria, which suggests the microbiota could not survive in the GIT. Probiotic candidate should therefore be tolerant to bile salt, gastric juices, and acid conditions. A previous study6 stated that the ability of a probiotic candidate could be measured through a tolerance assay to bile salt, gastric juices, and acid conditions. This study evaluates LAB and yeast from the colons of native chicken and Javanese duck, respectively, as probiotic candidates with resistance to bile salt and acid conditions, as well as inhibitory activities against pathogenic bacteria.
EXPRIMENTAL DETAILS Time and Location This experiment was conducted from July 2017 to November 2017 in the Laboratory of Microbiology and Mycology, Research Unit for Natural Product Technology (BPTBA), Indonesian Institute of Sciences (LIPI), Gunungkidul, Special Region of Yogyakarta.
Antimicrobial Activity Assay Lactic acid bacteria (LAB) were grown on de Mann, Rogosa, Sharpe (MRS) agar media (Oxoid) for 24 h at 37°C while yeast isolate was grown on yeast glucose chloramphenicol (YGC) agar media (Merck) for 24 h at 30°C. Both isolates were centrifuged at 4136 × g for 15 min at 4°C. Supernatant was neutralized by using 0.5 N NaOH (Merck) and sterilized by using millipore 0.20 μ. Inhibition activities against Escherichia coli FNCC 0194, Staphylococcus aureus FNCC 6049, Pseudomonas aeruginosa FNCC 0063, and Salmonella pullorum ATCC 13036 in nutrient agar (NA) media (Merck) were performed using agar diffusion methods7 with incubation for 24 h at 37°C . The experiment was arranged on a factorial design, which consisted of five treatments (crude bacteriocin from LAB AKK-30, yeast B-18, antibiotic penicillin, streptomycin, and erythromycin) and four pathogenic bacteria (E. coli, S. aureus, P. aeruginosa, and S. pullorum) and each treatment consisted of three replications. Twenty-five microliters of sterile supernatant were embedded in blank paper discs and placed in a plate containing pathogenic bacteria. LAB AKK-30 was isolated from the colon of native chicken8 and B-18 yeast was isolated from the colon of Javanese duck, which were preserved in the BPTBA microbiology laboratory.
Antibiotic Sensitivity Assay LAB and yeast isolate aged 24 h were inoculated on MRS agar media and YGC agar media. Antibiotic paper discs were put on agar media surfaces and then incubated for 24 h at 37°C (LAB) and 30°C (yeast). The clearing zone diameter around the paper disc was observed after the incubation time. The assay was measured by using the Kirby–Bauer method.9 The antibiotics were 10 μg penicillin G, 10 μg streptomycin, and 15 μg erythromycin. The experiment was arranged on a factorial design consisting of two isolates and three antibiotics with four replications.
Bile Salt Tolerant Assay The bile salt tolerant test was observed by a modified method10,11; 1 mL of LAB AKK-30 and yeast B-18 cultures aged 18 h were centrifuged at 4136 × g for 10 min at 4°C and washed twice using sterile phosphate buffered saline (PBS) (Oxoid). Pellets were added in 0.3 mL of PBS and mixed with 1 mL PBS containing 0.3% (w/v) bile salt (Merck). Each mixture was incubated for 3 h at 37°C for LAB and 30°C for yeast. Samples were observed at 0, 2, and 3 h. The viability was calculated by using serial dilution and plated on MRS agar media and YGC agar media with three replications.
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Gastric Juice Tolerant Assay Gastric juice tolerant assay referred to the modified gastric juice simulation.10,11 LAB culture was incubated on MRS broth (Conda) for 18 h at 37°C, while yeast culture was incubated on YGC broth (Conda) for 18 h at 30°C. From each culture, 1 mL was centrifuged at 4136 × g for 10 min at 4°C. Pellets were rinsed using sterile PBS twice and diluted in 0.3 mL PBS and mixed with 1 mL PBS pH 2 containing pepsin 0.3% (Sigma) (artificial gastric juice). LAB was incubated at 37°C, while yeast was incubated at 30°C for 45 min and both were sampled at 0, 15, and 45 min. Serial dilution was made on sterile PBS then inoculated on MRS agar media for LAB and YGC agar media for yeast for the viability test.
Acid Tolerant Assay Acid tolerant analysis was determined by the modified method of Torshizi et al. and Damayanti et al.10,11 LAB and yeast cultures aged 18 h were centrifuged at 4136 × g for 10 min at 4°C and the pellets were cleansed using sterile PBS twice and adulterated in sterile PBS before being inoculated on PBS pH 2 and pH 3. Cell viability was calculated by the total plate count (TPC) method on MRS agar media and YGC agar media, respectively.
Data Analysis Quantitative data of antibacterial activity and antibiotic sensitivity assays were analyzed by analysis of variance (ANOVA) followed by Duncan’s multiple range test to distinguish the effect of different treatment means using CoSTAT statistical software.12 Total of bacteria cells (cfu/mL) was converted to a logarithmic value and the viability percentage was calculated by dividing the total colonies after incubation (log10 cfu/mL) with the total colonies before incubation (log10 cfu/mL) and the results were multiplied by 100%.
RESULT AND DISCUSSION The current study uses two isolates, lactic acid bacteria (LAB), coded LAB AKK-30, and yeast, coded yeast B18. These were isolated from two different native poultry: LAB AKK-30 was isolated from the colon of a native chicken reared by a local farmer in Gunungkidul Regency; the bacteria were identified as Lactobacillus plantarum by biochemical identification.8 The similarity of LAB AKK-30 with L. plantarum was 92.3%. The second isolate, yeast B-18, was isolated from a Javanese duck’s colon preserved in the microbiology laboratory of BPTBA LIPI, and the morphology showed that the isolate was Saccharomyces cerevisiae (Fig.1).
(a)
(b) FIGURE 1. (a) Lactic acid bacteria AKK-30; (b) Yeast B-18.
Antibacterial activity assay using crude bacteriocin against pathogenic bacteria are shown in Table 1. Paper discs containing antibiotics were used as a positive control, while the samples with crude bacteriocin were from LAB AKK-300 and yeast B-18. The indicator of this assay was the diameter of the clearing zone, which indicates the ability of the crude bacteriocin to act against pathogenic bacteria. Both samples produced crude bacteriocin in different levels. There was a significant interaction (P < 0.01) between pathogenic bacteria and antibacterial
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substances. This means antibacterial substances had a specific inhibition against specific bacteria. Crude bacteriocin from the isolates had the ability to inhibit P. aeruginosa, S. aureus, and S. pullorum. Antibacterial substances from yeast B-18 tended to inhibit those bacteria more than crude bacteriocin from AKK-30. This result could be influenced by bactericidal activities of the bacteriocin. LAB AKK-30 produced lactic acid, while yeast B-18 produced oxylipin and these bacteriocin might have different abilities against pathogenic bacteria.13 The mechanism of antibacterial inhibition occurred in two stages: first was bacteriocin absorption on specific and nonspecific receptors of the pathogenic bacterial membrane cells and the crude bacteriocin became sensitive mainly to proteolytic enzyme; the second stage was irreversible and involved lethal changes in sensitive strains.14 TABLE 1. Antibacterial activity of antibiotics, LAB AKK-30 and yeast B-18 isolates
Antibacterial Substances Streptomycin 10 μg Penicillin 10 μg Erythromycin 15 μg Yeast B-18 LAB AKK-30
P. aeruginosa 11.19±2.61F 15.06±3.18BCD 17.69±0.63B 15.46±1.60BCD 9.84±2.98EFG
Clearing Zone Diameter (mm) S. aureus E. coli 11.20±0.46F 6.18±0.93G 13.34±0.23BCDE 0.00±0.00H A 27.52±0.65 9.97±0.88EFG B 17.82±2.59 0.00±0.00H 6.44±3.13G 0.00±0.00H
S. pullorum 13.01±0.16CDE 16.76±3.49BC 11.90±2.15DEF 15.04±2.37BCD 7.29±0.96FG
Note: Means in the same column and row differ significantly (P < 0.01) The susceptibilities of the isolates to antibiotics are shown in Table 2. The results indicate that yeast B-18 had no clearing zone after 24 h incubation, while the addition of penicillin and erythromycin on LAB AKK-30 media showed different diameter clearing zones. Clearing zones do not always indicate that the microorganism is sensitive to antibiotic. Stanley et al.15 stated that the Lactobacillus strain isolated from chickens possessed high antibiotic resistance. There were three classifications of microorganism resistance: resistance (R), intermediate (I), and sensitive (S). These classifications are based on the clearing zone diameter, and each antibiotic had a different range of clearing zone. Streptomycin grouped (R) with a clearing zone diameter ≤ 11 mm, (I) with a clearing zone diameter 12–14 mm, and (S) with a clearing zone diameter ≥ 15 mm; penicillin is categorized (R) with a clearing zone diameter ≤ 28 mm and (S) with a clearing zone diameter ≥ 29 mm; and erythromycin is classified (R) with a clearing zone diameter ≤ 13 mm, (I) with a clearing zone diameter 14–22 mm, and (S) with a clearing zone diameter ≥ 23 mm. These results were close to the study by Stanley et al.15 which stated that LAB (Lactobacillus strain) had low susceptibility to antibiotics. In a previous study, three selected broiler chicken–indigenous LAB had some degree of antibiotic resistance against several tested antibiotics.10 The resistance of yeast isolate to antibiotics made it suitable for animals undergoing antibiotic treatment and potentially more profitable than bacteria for therapeutic use.16 TABLE 2. Antibiotic sensitivity assay of LAB AKK-30 and yeast B-18 isolated from native chicken and Javanese Duck gastrointestinal tract Clearing Zone Diameter (mm) Antibiotic Yeast B-18 LAB AKK-30 Streptomycin 10 μg 0.00±0.00 (R) 0.00±0.00 (R) Penicillin 10 μg 0.00±0.00 (R) 23.11±0.73 (R) Erythromycin 15 μg 0.00±0.00 (R) 24.18±1.04 (S) Note: Means in the same column and row with different superscript differ significantly P < 0.01. R: resistant; S: sensitive.
In order to provide health benefits to the host, one of the main characteristics a probiotic must have is resistance to the environment of the GIT, including acid and bile salt.17 The tolerant assay of bile acid, gastric juice, and pH acid results are shown in Table 3. The LAB isolate had the ability to survive on bile salt after 3 h incubation; it would be possible to de-conjugate bile salt and the LAB isolate might be effective to reduce serum cholesterol in poultry. Bile salt tolerance in the GIT is associated with bile salt hydrolase (BSH) activity.18 BSH broke down the peptide linkage of bile acids, which removed the amino acid group from the steroid core; the unconjugated bile acids precipitate at low pH.19 The viability percentage of yeast B-18 tends to decrease after 3 h incubation. This result contradicted Fakruddin et al.20 which reported that yeast could survive at high bile concentrations. Acid tolerant assay for yeast B-18 showed a tendency to increase the number of colonies, as seen in the increase of colonies formed after a relatively short incubation time. In the gastric acid tolerance test, isolate viability reached 104.62% at min 15 and increased at min 45. Similar results occurred in the acid tolerant assay at pH 3. Cell viability increased to 118.34% after 45 min incubation then decreased slightly after 90 min incubation to 98.71%. In the acid tolerant assay at pH 2, yeast viability decreased significantly to 3.39% after incubation of 90 min. These results show that yeast B-18 was better able to survive in pH 3 conditions than pH 2. Syal and Vohra21 reported that Saccharomyces cerevisiae isolated from fermented food could grow well under acidic conditions at pH 2, 2.5, and 3. The ability of isolates, which differed from previous studies, might be caused by the
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different sources of the isolates. In this study, the isolates of yeast B-18 were from Javanese duck colon and were more tolerant of pH 3 than pH 2. TABLE 3. Viability of LAB AKK-30 and yeast B-18 on bile salt and gastric juice resistance Isolates Percentage of Viability (%) Time LAB AKK-30 Yeast B-18 0 min 100.00±0.00A 100.00±0.00A Bile Salt Viability 120 min 82.41±31.07A 23.91±4.63B A 180 min 135.51±69.39 12.07±1.88B 0 min 100.00±0.00A 100.00±0.00A Gastric Juice Viability 15 min 17.90±1.26B 104.62±7.28A 45 min 0.01±0.00C TNTC* 0 min 100.00±0.00A 100.00±0.00A pH 2 Viability 45 min 0.37±0.39D 38.66±2.48B 90 min 0.08±0.13D 3.39±0.48C 0 min 100.00±0.00B 100.00±0.00B pH 3 Viability 45 min 0.26±0.15C 118.34±15.43A 90 min 0.30±0.43C 98.71±7.70B Note: Means in the same row with different superscript differ significantly P < 0.01. *Data from the cell viability after 45 min incubation was not included in average calculation and statistical analysis, TNTC: Too Numerous To Count
In the gastric juice tolerant assay, the cell viability of the LAB isolate decreased, while the yeast isolate survived after 45 min incubation. This result was in line with Damayanti et al. and Fakruddin et al.20,22 Moreover, Fakruddin et al.20 stated that S. cerevisiae IFST062013 had the ability to grow in media containing pepsin as the artificial gastric juice. The ability of the microorganism to survive in acid medium was influenced by many factors, including temperature, pH, nutrient ability, and previous natural habits.23 The current study suggests that isolate viability was affected by similar factors. The different sources of the isolates might have caused different mechanisms in using nutrients based on the current location and previous natural habitat. Low extreme pH might be a suitable environment for yeast, whereas LAB had to survive in acid conditions. The adaptability of microbiota in different environmental conditions closely affected growth and survivability.24
CONCLUSION LAB AKK-30 and yeast B-18 isolated from the colons of native chicken and Javanese duck, respectively, had bactericidal activity against P. aeruginosa, S. aureus, and S. pullorum. Yeast B-18 showed resistance to antibiotics such as streptomycin, penicillin and erythromycin, while LAB AKK-30 was only resistant to streptomycin and penicillin. In acid conditions, yeast B-18 had a higher viability percentage than LAB AKK-30. LAB isolate was more resistant to bile salt, which means both isolates can survive in the GIT. LAB AKK-30 and yeast B-18 have potential as probiotic candidates and need to be encapsulated for direct use in poultry production.
ACKNOWLEDGEMENT This research was held by the research collaboration between Laboratory of Feed Science and Technology, Department of Nutrition and Feed Technology, Faculty of Animal Sciences, Bogor Agricultural University (IPB) and Indonesian Institute of Sciences (LIPI). We appreciate great thankfully to Madina Nurohmah, S.Pt. for supporting this research. The first author (R.S.H. Martin) would thank to Indonesian Ministry of Education and Culture (Kemendikbud) for Master Scholarship Program (Beasiswa Unggulan Masyarakat Berprestasi).
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