Evaluation of cellulolytic Bacillus isolates as animal probiotics and the effect of B. glycinifermentans SK 4275 on in-vitro fermentation of elephant grass

CONTENTS

List of Table

List of Figure

List of Abbreviation

Abstract

Chapter 1. Introduction
1.1. Elephant grass as a feed resource for ruminants
1.2. Concerns of antibiotics as feed additives
1.3. The role of Bacillus strains as animal probiotics
1.4. Bacillus glycinilermentans and B. paralichinelbrmis
1.5. Purpose of this study

Chapter 2. Material and Methods
2.1. Collection and preparation of elephant grass
2.2. Isolation of Bacillus strains from elephant grass
2.3. Screening for enzyme activity
2.4. Screening for probiotic properties of B. glycinilermentans SK4275 and B. Paralichenilbrmis SK4278 in vitro
2.4.1. Acid tolerance
2.4.2. Bile tolerance
2.4.3. Sodium chloride tolerance
2.4.4. Antimicrobial activity
2.4.5. Antibiotic sensitivity
2.4.6. Hemolytic activity
2.5. In vitro ruminal fermentation
2.5.1. Substrate, Bacillus and treatments
2.5.2. In vitro study
2.5.3. Sampling and analysis
2.6. Statistical analysis

Chapter 3 Results and discussion
3.1. Identification of isolated Bacillus strains
3.2. Enzyme activity
3.3. In vitro assessment of characteristics for survival in gastrointestinal tract
3.3.1. Acid tolerance
3.3.2. Bile tolerance
3.3.3. Antimicrobial activity
3.3.4. Sodium chloride tolerance
3.4. Biosafety assessment
3.4.1. Antibiotic sensitivity
3.4.2. Hemolytic activity
3.5. In vitro fermentation characteristics
3.5.1. pH, total gas and ammonia-N concentration
3.5.2. Volatile fatty acids
3.5.3. Total viable count in incubation fluid
3.5.4. In vitro NDF digestibility and ADF digestibility
3.6. Discussion of in vitro fermentation findings

Chapter 4. Conclusion

References

Abstract (In Korean)

List of Table

Table 1. Chemical composition of elephant grass (P purpureum) sample before fermentation

Table 2. Chemical composition of McDougaU’s buffer

Table 3. Description of Bacillus strains isolated from elephant grass

Table 4. Acid resistance (synthetic gastric juice, pH 2.5) of Bacillus strains isolated from elephant grass

Table 5. Bile tolerance (0.3% oxygall) of Bacillus strains isolated from elephant grass

Table 6. Sodium chloride (NaCl) tolerance of Bacillus strains isolated from elephant grass

Table 7. Susceptibility profile of Bacillus strains to antibiotics

Table 8. Effect of B. glyciniíbrmentans SK4275 addition on in vitro pH, ammonia-N concentration and total gas production of elephant grass

Table 9. Effect of B. glyciniíbrmentans SK4275 addition on volatile fatty acids concentration of in vitro incubation fluids of elephant grass

Table 10. Effect of B. glyciniíbrmentans SK4275 addition on total VFAs and acetate to propionate ratio

Table 11. Effect of B. glyciniíbrmentans SK4275 addition on total viable bacteria of in vitro incubation fluids of elephant grass

Table 12. Effect of B. glyciniíbrmentans SK4275 addition on in vitro NDF digestibility and ADF digestibility of elephant grass

List of Figure

Figure 1. Elephant grass (Pennisetum purpureum)

Figure 2. Enzyme activity of Bacillus isolates

Figure 3. Antimicrobial activity of B. glycinifermentans SK against pathogens

Figure 4. Antimicrobial activity of B. paralichenilbrinis SK against pathogens

Figure 5. Antibiotic sensitivity of B. glycinifermentans SK4275

Figure 6. Antibiotic sensitivity of B. paralichenilbrmis SK4278 .

Figure 7. Hemolytic activity of Bacillus strains

List of Abbreviation

Abbildung in dieser Leseprobe nicht enthalten

Abstract

Evaluation of cellulolytic Bacillus isolates as animal probiotics and the effect of B. glycinifermentans SK 4275 on in vitro fermentation of elephant grass

David, Nsubuga

Department of Animal Science and Technology Graduate School of Konkuk University

In this study two Bacillus strains Bacillus glycinilermentans SK4275 and Bacillus paralichinelbrmis SK4278 were isolated from elephant grass (Pennisetum purpureum) and identified by 16S rRNA gene sequence analysis. The isolates were examined for their cellulolytic enzyme activity, sodium chloride tolerance and in addition to that, their in vitro probiotic properties such as acid tolerance, bile tolerance, antimicrobial activity and biosafety were also determined. The effect of live Bacillus glycinilermentans SK 4275 on the in vitro fermentation and digestibility of elephant grass was investigated by adding the test Bacillus strain at three doses (control [without addition of Bacillus\, TI; (lxlO8 CFU (colony-forming-units)) and T2; (lxlO10 CFU). According to the results B. glycinilermentans SK4275 showed significant cellulolytic enzyme activity for cellulase, amylase, and xylanase compared to B. paralichenilbrmis SK4278 that showed lesser ability to produce enzymes. Both B. glycinilermentans SK4275 and B. paralicheniformis SK4278 showed potential probiotic characteristics and sodium chloride tolerance up to 10% (w/v) NaCl and 8% (w/v) NaCl respectively. Both test strains exhibited no antagonist activity against any of the indicator pathogens (Shigella dysenteriae SK4192, Escherichia marmotae SK4193, Escherichia Coli SK4195, Enterococcus laecalis SK4198, Shigella üexneri SK4264 and Salmonella pullorum SK3360) tested. On the other hand, both B. glycinilermentans SK4275 and B. paralichinelbrmis SK4278 varyingly tolerated bile salt (0.3% Oxygall), and survived in acid (synthetic gastric juice pH 2.5). Furthermore the strains met the safety criteria for selection of useful probiotics in feed including susceptibility to antibiotics and exhibited no hemolytic activity. Results of in vitro fermentation showed that, compared to control the addition of B. glycinilermentans SK4275 in fermentation sample did not significantly affect pH, concentration of NH3-N, total gas, in vitro NDF and in vitro ADF digestibility of elephant grass for samples drawn between 0 to 12 hours. The pH measured in ruminal fluid was significantly increased (p<0.05) with addition of Bacillus compared to the control at 24 and 48 hours. Also the total gas measured was significantly higher in fermentation samples containing Bacillus culture (T1 and T2) than in control (p<0.05) especially at 24 h and 48h. The concentration of NH3-N in the incubation fluids was significantly increased (p<0.05) with addition of Bacillus (at 48 hours) compared to control. Furthermore, concentration of acetate, butyrate, valerate, isobutyrate, and isovalerate were significantly decreased (p<0.05) in rumen fluids containing T2 (after 24 h) when compared to T1 and control while concentration of total volatile acids did not significantly differ (p<0.05) between treatments except at 12 h and 24 h Bacillus at dose lxlO10 CFU (T2) significantly (p<0.05) decreased the total volatile fatty acids concentration when compared to T1 and control. Acetate to propionate ratio was significantly (p<0.05) decreased in treatments containing Bacillus at dose lxlO10 CFU (T2) at 9 h and 48 h compared to T1 and control. The total number of bacteria counted on both Luria Bertani (LB) and Yeast Mold (YM) agar plates were numerically or significantly (p<0.05) increased in ruminal fluids containing Bacillus when compared to control. Addition of Bacillus significantly increased (p<0.05) in vitro NDF digestibility and in vitro ADF digestibility of the substrate at 24h and 48h. The results indicated that B. glycinifsrmentans SK4275 could be a potentially useful probiotic additive for improving in vitro fermentation and digestion of elephant grass. Therefore the present study proposes the possibility of combining the probiotic attributes of a Bacillus strain with its cellulose degrading ability to improve rumen fermentation of animal feed.

Key words: Bacillus glycinifsrmentans, probiotics properties, elephant grass, enzyme activity, in vitro fermentation

Chapter 1. Introduction

1.1. Elephant grass as a feed resource for ruminants

Elephant grass (Pennisetum purpureum) also known as napier grass, is a major livestock feed source for small holder dairy production systems in Uganda (Kabirizi et al. 2013). Elephant grass highly prefferd among forage grasses due to its drought tolerance and high yeilding potentials. However, the presence of structural cell wall carbohydrates i.e., cellulose and hemicellulose in P. purpureum makes this grass have low-degestibility in ruminants which is the major limitation to utilization of most forage (Van Soest, 1994). According to Hatfield et al., (1999) the plant cell wall is the main energy source for cattle despite the fact that less than a half of forage ingested is efficiently digested and utilized by ruminants. Yet animal performance is directly related to the feed digestibility and microbial fermentation in the rumen (Beever, 1997). The demand for improved animal productivity and enhanced feed digestibility is increasing and such demand can be achieved through feeding diets containing probiotics (Manhar et al. 2015).

Abbildung in dieser Leseprobe nicht enthalten

Figure 1. Elephant grass (Pennisetum purpureum)

1.2. Concerns of antibiotics as feed additives

Although the use of antibiotics as a feed additive greatly contributed to improving growth performance and controlling disease in animals, their overuse has led to development of bacterial resistance to antibiotics and residues in animal products (Chen et al., 2009). The resulting public and scientific concerns prompted countries such as south korea and the european union to ban the use of antibiotics as feed supplements (Phillips, 2007; Chen et al., 2009; Lee et al., 2011). Consequently, research aimed at developing alternatives to antibiotics has received considerable attention (Turner et al., 2001). Probiotics have been considered as potential alternatives to antibiotics.

1.3. The role of Bacillus strains as animal probiotics

Probiotics are viable microbial feed additives which when administered may improve the intestinal microbial balance of a host animal (Fuller 1989). Generally high cellulose content of plant feeds can be hydrolyzed by cellulolytic probiotics in addition to rumen microbes thus providing a source of energy to the animal rather than losing feed in the undigested feces (Manhar et al. 2015). The use of Bacillus strains as probiotic additives for enhancing rumen fermentation of fibrious animal feed has attracted increasing interest among researchers.

Bacillus species can generate dormant exogenous spores resistant to heat, desiccation, enzymatic degradation, and the gastric acidic conditions (Hong et al., 2005, Leser et al., 2008), produce extracellular enzymes that can enhance feed digestibility, in addition to stimulating the immune system of host animal (Sun et al., 2010), thus improving growth performance, feed conversion ratio (Sun et al., 2010; Zhou et al., 2010). Balcazar et al. (2006) and Verschuere et al. (2000) reported that probiotic supplementation resulted on the following host benefits such as inhibiting pathogens, enhancing immune response, providing nutrients and enzymes that aid digestion in the host. In another study Manhar et al. (2015) Bacillus amyloliqueiaciens AMS showed desirable probiotic characteristics in addition to cellulolytic activity in vitro. Those results suggested that enzymes produced by Bacillus can transform complex molecules such as ligno-celluloses which are a limiting factor in forage, into simpler molecules (Wizna et al., 2009).

Bacillus glycinifermentans have also been reported to have ability to grow in bile media which makes it a potential probiotic strain for use livestock productivity (Stadermann et al., 2017).

1.4. Bacillus glycinifermentans and B. paralichineformis

There has been recent reclassification within the genus Bacillus leading to description of novel species for example Bacillus gylcinifermentans (Kim et al., 2015) and B. paralichenifermis (Dunlap et al., 2015).

Dunlap et al. (2015) and Kim et al. (2015), isolated the two novel bacterial isolates from soybean fermented paste and were biochemically characterized as gram positive, anaerobic, motile, rod shaped endospore-forming bacteria. Furthermore, 16 rRNA gene analysis indicated that these Bacillus strains are closely related to B. sonoresis and B. lichenilbrmis.

Furthermore, Stadermann et al. (2017) reported that B. glycinifermentans possess gene clusters responsible for secondary metabolites such as bacitracin which may inhibit pathogens in the host. Recently attempts have been made to manupilate rumen digestion process and improve effeiciency of forage utilisation in ruminants. Manhar et al. (2016) tested the possibility of utilizing probiotic Bacillus strains to degrade cellulose in animal feed thereby enhancing digestibility and animal production.

1.5. Purpose of this study

Not all Bacillus strains are equally resistant to the harsh evironment in the gastrointestial tract besides there are also variations in enzyme activity, and antimicrobial activity, hence selection of appropriate Bacillus strains is essential for the effectiveness of probiotic additives for use in animal feed (Guo et al., 2006). The purpose of this study was to describe in vitro probiotic properties such as acid tolerance, bile tolerance, antimicrobial activity and safety, of two Bacillus strains, B. glycinifermentans SK4275 and B. paralichenilbrmis SK4278 isolated from elephant grass in addition to their cellulolytic enzyme activity. The effect of live B. glycinilermentans SK 4275 on in vitro fermentation of elephant grass (Pennisetum purpureum) was also investigated.

Chapter 2. Material and Methods

2.1. Collection and preparation of elephant grass

Elephant Grass (Pennisetum purpureum ‘NARO PI’) was grown at the national livestock resources research institute, Nakyesasa in Uganda. The grass was harvested at 60 days and its leaves were chopped into small parts of 2-3 cm followed by drying in oven at about 55 °C for 48-72 hours. Dried grass sample was then ground and sieved through a 1 mm screen before being sent to Konkuk University, Korea for subsequent in vitro experiments.

2.2. Isolation of Bacillus strains from elephant grass

The isolation of bacterial strains was done according to methods described by Kunchala et al. (2016) with modifications. 10 g of elephant grass sample was added with 90 ml of sterile distilled water in a conical flask and incubated at 37.2 °C for 72 hours at 200 rpm. Then 0.1 ml of the sample was added to 0.9 ml sterile physiological saline (0.85 g of NaCl in 1L of water). After through mixing serial dilution was made up to 106 dilutions in physiological saline. Then 0.1 ml of the dilutions 105 and 106 was poured on nutrient agar plate and spread out. The plates were incubated at 37 °C for 24 h. The bacterial colonies grown on the plates were purified by further streaking on Luria bertani (LB) agar plates. The pure colonies were identified by 16s rRNA gene sequence analysis and stocked in deep-freezer (MDF-U53V, SANYO, Japan) at -70°C in LB broth containing 10% of DMSO (Dimethylsulfoxide! Georgiachem).

2.3. Screening for enzyme activity

LB agar was prepared by mixing 25 g of LB broth (Tryptone-10 g, yeast extract-5 g, NaCl-10 g per liter) with 20 g of bacto-agar (BD science) in 1000 ml of distilled water in a flask. 1% of each carboxy-methyl-cellulose, soluble starch, skim milk and xylan were added into the LB agar to test for cellulase, amylase, protease, and xylanase production respectively. 1% of tributyltin was added in spirit blue agar to test for lipase production. The agar media were mixed thoroughly and autoclaved for 15 minutes at 121 °C. After cooling the medium were poured into petri-dishes and allowed to solidify over 2 days. Pure colonies of the test isolates were streaked on respective enzyme media and incubated at 37 °C for 72 hours. The plates tested for amylase, cellulase and xylanase were flooded with 1% gram’s iodine (5 ml) and iodine poured off after 3 minutes. Appearance of a clear zone around the colonies grown on agar plates indicated that the test isolate was positive for respective enzyme activity.

2.4. Screening for probiotic properties of B. glycinifermentans SK4275 and B. paralicheniformis SK4278 in vitro

The bacterial cultures stored at -70 °C were revived by culturing on LB agar plates for at 37°C for 24 hours. Single colony from the incubated plates were selected and inoculated in a tube containing LB broth (5 ml) and cultured at 37 °C for 24 h in a shaking incubator (VS-8480SF, Vision scientific, Korea).

2.4.1. Acid tolerance

The method previously described by Kim et al. (2007) was used to examine the survival of the Bacillus isolates under synthetic gastric conditions. Bacillus strains (B. glycinilermentans SK4275 and B. paralichenilbrmis SK4278) were each cultured in LB broth at 37 °C for 16 hours. The cells were centrifuged for 10 minutes at 10,000 rpm, 4°C, followed by washing the cells with phosphate buffered saline (PBS: pH 7.4, 0.144% Na2HP04, 0.5% NaCl, 0.024% KH2PO4) thrice. Following washing the cells were re-suspended in 1000 gl of PBS (adjusted to about 108 CFU/ml). 0.1 ml of cells was added to 0.9 ml of synthetic gastric juice consisting of 0.5% NaCl, 0.1 peptone, and 0.3% pepsin (BD Science, USA). The pH adjusted to 2.5 using hydrochloric acid and culture examined after 30 , 60 and 180 minutes. The viable cells were enumerated by spotting 10-fold dilutions of the culture on LB agar plates and incubating at 37 °C for overnight. The survival rate of the Bacillus strains was calculated percentage of colonies grown on 37 °C agar compared to the colonies observed at 0 h.

2.4.2. Bile tolerance

The bile tolerance was done following the methods by Kheadr et al., (2007) with some modifications. Colonies of each of the bacterial strains were grown in LB broth at 37 °C for 24 hours. 10 ml of the cultures was centrifuged (5,000 x g, 20 min, 4°C), and cells were washed with PBS. Then 0.1 ml of cultured broth was added to fresh LB broth containing 0.3% (w/v) Oxgall (BD science). Samples were taken after standing for 0, 0.5, 2, and 3 hours, serially diluted in PBS followed by plating on LB agar using Spot-lawn method and incubating for 24 hours at 37 °C. After the incubation, the survival rate was calculated as the percentage of Bacillus colonies grown on LB agar compared to number of colonies at 0 hour. Three tests were performed for each Bacillus strain.

2.4.3. Sodium chloride tolerance

Isolated test Bacillus colonies were streaked on LB agar plates amended with 2, 4, 6, 8 and 10% sodium chloride. After 24 hours incubation at 37 °C, tolerance to sodium chloride was determined by observing the growth of each strain on the different sodium chloride treated agar plates. The tests were performed in triplicate.

2.4.4. Antimicrobial activity

Six pathogens used in this study including Shigella dysenteriae SK4192, Escherichia marmotae SK4193, Escherichia coli SK4195, Enterococcus íaecalis SK4198, Shigella üexneri SK4264 and Salmonella pullorum SK3360. Antimicrobial activity of the Bacillus strains against the pathogens was determined according to the agar well diffusion method described by Hernandez et al. (2005). The Bacillus isolates as well as indicator pathogens were cultured separately in LB (Luria-Bertani; Difco, France) broth at 37 °C for 24 hours. The overnight grown pathogen cultures were uniformly spread onto the surface of LB plates with the help of sterile cotton swabs. Sterile pasteur tube was used to cut 6 mm diameter wells from the agar plate. The wells were poured with 0.1 ml of supernatant prepared by centrifuging the Bacillus culture at 8000 rpm for 15 min. The plates were incubated at 37 °C for 24 h. The diameter of clear zone around each well was measured.

2.4.5. Antibiotic sensitivity

The antibiotic resistance pattern of Bacillus strains was examined using nine antibiotic paper discs including cefepime, gentamycin, vancomycin, ampicillin, tetracycline, oxacillin, ciprofloxacin, chloramphenicol, and clindamycin (Oxoid, UK). Paper - disk method as described by Zhou et al, (2005) was used to perform antibiotic sensitivity test. The isolates were inoculated and cultured overnight. A sterile cotton bud was used to spread Bacillus isolates evenly on the surface of LB agar plates. Antibiotic paper disc (6 mm diameter) was positioned on the surface agar plate with the aid of sterile forceps and all plates were incubated overnight at 37 °C. Antibiotic sensitivity of each Bacillus strain was assessed by measuring the diameter the clear zone surrounding the paper disc.

2.4.6. Hemolytic activity

Hemolysis was determined as previously detailed in the method by Anand et al. (2000) with modifications. Briefly the test Bacillus strains were streaked on blood agar (5% v/v sheep blood) plate. After incubation (for 72 h at 37 °C) plates were observed for color change of blood agar. Formation of a transparent or clean hemolytic zone on the blood agar indicates ^-hemolysis and brownish or greenish hemolytic zone indicates a-hemolysis. Where no such zone (no color change on the blood agar) around the Bacillus colonies is indicates non-hemolytic activity (y-hemolysis).

2.5. In vitro ruminal fermentation

2.5.1. Substrate, Bacillus and treatments

Elephant Grass (P. purpureum) was used as the substrate in in vitro fermentation experiment. Elephant grass was grown in Uganda, dried at 65°C and ground through a 1 mm screen as explained earlier. The chemical content of the elephant grass sample is shown in table 1.

The effect of live Bacillus strain, Bacillus glycinifsrmentans SK 4275 on the in vitro fermentation of elephant grass was conducted by adding the Bacillus strain at three doses (CON; 0 CFU [without addition of Bacillus\, TI; lxlO8 CFU, T2; lxlO10 CFU)

Rumen fluid was donated by a rumen-cannulated Holstein cow (650 ± 30 kg) raised at Konkuk university farm, Chung-ju. The cow was fed on total mixed ration twice a day with water provided ad libitum. Rumen fluid was collected at 08.00 hours (before morning feeding). The rumen culture was deposited in a pre-warmed flask and immediately taken to the laboratory.

Table 1. Chemical composition of elephant grass (P. purpureum) sample before fermentation

Abbildung in dieser Leseprobe nicht enthalten

2.5.2. In vitro study

In vitro incubation was conducted according to methods of Tilley and Terry (1963). Rumen fluid was seeped through four layers of surgical gauze to remove feed particles. About 500 ml of rumen fluid was mixed with 2000 ml of prepared McDougall’s buffer (Table 2). Oxygen-free carbon dioxide gas (CO2) was bubbled through the rumen fluid-buffer mixture for 30 minutes at 39 °C. Approximately 300 mg of grass sample was measured into 60 ml serum bottles followed by addition of B. glycinilbrmentans SK 4275 at doses mentioned above.

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