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Dairy propionibacteria as direct-fed microbials: in vitro effect on acid metabolism of Streptococcus bovis and Megasphaera elsdenii

Dairy propionibacteria as direct-fed microbials: in vitro effect on acid metabolism of... Ruminal acidosis caused by accumulation of lactic acid, a decrease of pH in the rumen and subsequent imbalance of the rumen fermentation process, affects the health and productivity of dairy cows and beef cattle. Direct-fed microbials have potential for use in the control and prevention of ruminal acidosis. This study investigated the interaction between five strains of dairy propionibacteria, Megasphaera elsdenii and Streptococcus bovis in various co-culture combinations in a simulated rumen environment comprising unmodified rumen digesta supplemented with excess glucose. While suppression of lactic acid accu- mulation by both the dairy propionibacteria and M. elsdenii in the presence of S. bovis in the simulated rumen conditions was evident, propionibacteria were found to be more effective than M. elsdenii in controlling lactic acid levels. . . . Keywords Probiotics Propionibacteria Rumen acidosis Rumen microbes Findings haboring a variety of microorganisms, some of which have been shown to influence the development of acidosis. Propionibacteria are characterized by utilization of lactic acid Streptococcus bovis is an inhabitant of the rumen environ- as the favored carbon source, with propionic acid produced as ment, but is usually found in relatively low numbers in the a by-product (Luo et al. 2017a). Dairy propionibacteria have healthy rumen. However, S. bovis is relatively acid tolerant been proposed as potential probiotic candidates for the treat- (Russell and Hino 1985; Miwa et al. 2000) and has been ment and prevention of ruminal acidosis—a prevalent disor- identified as the major lactic acid producer responsible for der in ruminants (Luo et al. 2017b). Ruminal acidosis is the development of acidosis (Maroune and Bartos 1987; caused primarily by the inclusion of a high percentage of Owens et al. 1998;Enemark 2008). Although antibiotic treat- readily fermentable dietary carbohydrates. This disorder pre- ment has proven to be effective in treating this condition, sents as an accumulation of lactic acid with a decrease of pH in potential facilitation of the spread of antibiotic-resistant bac- the rumen and subsequent imbalance of the rumen flora and teria makes this an unattractive option for routine use in fermentation processes, resulting in impaired health and pro- preventing acidosis (Millet and Maertens 2011). Therefore, ductivity of dairy cows and feedlot beef cattle (Enemark 2008; alternative approaches such as use of direct-fed microbials Luo et al. 2017a, b). The rumen is a complex environment (DFM) to control the growth of S. bovis are becoming increas- ingly popular (Luo et al. 2017b). Megasphaera elsdenii, the major lactic acid utilizer in the rumen, is able to use lactate, fructose and glucose as carbon * Chaminda Senaka Ranadheera sources, and produces propionate, acetate and butyrate as ma- senakar@email.com jor metabolic products (Holt et al. 1994). In the healthy rumen, M. elsdenii mainly utilizes the maltose hydrolyzed from starch School of Environmental and Life Sciences, The University of and the lactate produced by S.bovis as carbon sources (Russell Newcastle, Callaghan, NSW 2308, Australia et al. 1981;Hino et al. 1994), and, therefore, maintains the School of Agriculture and Food, Faculty of Veterinary and lactic acid level. However, this balance is often disrupted when Agricultural Sciences, The University of Melbourne, Melbourne, VIC 3010, Australia cattle are fed concentrates that include a higher percentage of starch, because it stimulates the growth of amylolytic bacteria School of Health Sciences, The University of Newcastle, to produce more volatile fatty acids (VFAs) and glucose. Since Callaghan, NSW 2308, Australia 154 Ann Microbiol (2018) 68:153–158 S. bovis can use both starch and glucose as carbon sources, this 2017b). Maximum grown (~10 cfu/ml) and saline-washed bac- increasing availability of substrate stimulates the rapid growth terial preparations of propionibacteria, S. bovis and M. elsdenii of S. bovis and leads to accumulation of lactic acid causing were used as inoculants. The five strains of dairy acidification in the rumen (Hino et al. 1994). The effects are propionibacteria used in the study, along with the abbreviated further exacerbated by the vulnerability of M. elsdenii to acidic names in brackets used throughout this paper, were conditions (Russell and Dombrowski 1980), which reduces Propionibacterium jensenii 702 (PJ702), P. acidopropionici their capacity for lactic acid consumption. ATCC 25562 (PA25562), P. acidopropionici 341 (PA341), Although propionibacteria are common inhabitants in the P. freudenreichii CSCC 2206 (PF2206) and P. freudenreichii rumen, they are normally present in low numbers (Oshio et al. CSCC 2207 (PF2207). There were ten preparations assigned 1987) and therefore exert no significant influence in control- in this study, both as two-strain and three-strain co-cultures. ling the development of acidosis. However, it has been dem- The details of the inoculation for each preparation are listed in onstrated that the introduction of dairy propionibacteria to a Table 1. Both two- and three-strain co-culture studies were per- nutrient broth medium containing co-cultures of S. bovis and formed. No additional bacterium was inoculated to the control. M. elsdenii was able to influence the fermentation process as For the treatment SB, only one strain of bacteria (S. bovis)was the lactic acid was efficiently converted into acetic acid and introduced. Two strains of bacteria were inoculated to treat- propionic acid (Luo et al. 2017b). These VFAs produced by ments PJ702 + SB, PA25562 + SB, PA341 + SB, PF2206 + dairy propionibacteria can then be absorbed through the rumen SB, PF2207 + SB and ME+SB, which contain S. bovis and wall and serve as an energy source for cattle (Dieho et al. either one strain of Propionibacterium or M. elsdenii.Three 2016). Although these findings indicated that the dairy different strains of bacteria S. bovis, P. jensenii 702 and propionibacteria may have potential in the alleviation of rumi- M. elsdenii were inoculated together for the treatment nal acidosis, given the complexity and the diversity of healthy PJ702 + ME+SB. All inoculated rumen samples were incubated ruminal micro-flora, the influence of other indigenous micro- in a CO incubator (Thermo Electrone, Thermo Scientific, flora on these interactions in the same growth environment Waltham, MA) with 10% CO under 37 °C for 48 h. At 0 h, must also be considered. In relation to this, the interaction 2 h, 4 h, 8 h, 12 h, 24 h and 48 h post inoculation, 5 ml sample between dairy propionibacteria, S. bovis,and M. elsdenii in was taken for high pressure liquid chromatography (HPLC) the rumen environment has not been well documented. analysis for the lactic, propionic, and acetic acid profiles as Hence, this study was designed to examine the metabolic in- described previously (Luo et al. (2017a) using a HPLC system teractions between dairy propionibacteria, S. bovis and (Hewlett-Packard 1100 DAD, Santa Clara, CA) fitted with a M. elsdenii in unmodified rumen fluid. In this context, the Pyrospher RP-18 (125 mm × 4 mm, 5 μm) column (Hewlett- introduction of glucose and S. bovis to the rumen content Packard). Bacterial cell abundances were not quantified during samples was carried out to create in vitro conditions similar analysis. Comparisons of the difference between the acid con- to those of ruminal acidosis. The hypothesis was that, under centration curves against time in different preparations in rumen the simulated ruminal acidosis conditions, the inoculation of contents were performed using the General Linear Model either propionibacteria, M. elsdenii, or their combination, Repeated Measurement in SPSS (PASW statistic 18), to mea- would prevent the accumulation of lactic acid via consumption sure the difference between the trajectories of each individual and conversion to acetic and propionic acids. Moreover, that acid concentration curve across the whole experimental period. the extent of the effect would vary dependent upon the specific In this study, the acid concentration profiles were altered propionibacteria strains involved. significantly by the introduction of different strains of Whole rumens (beef cattle) were obtained post-mortem from Propionibacterium and M. elsdenii in the glucose-fortified ru- a local abattoir (Kurri Meats, Newcastle, Australia) as part of the men content samples inoculated with S. bovis (Fig. 1). Among waste by-product of normal abattoir operations. In accordance the tested propionibacteria, PA341 and PJ702 were the strains with Australian government guidelines for the use of animals found to be associated with the lowest levels of lactic acid for scientific purposes, the study was exempt from ethical ap- accumulation and highest production of acetic and propionic proval requirements on the basis that no live animals were han- acid. By comparison, the PA25562, PF2206, and PF2207 treat- dled, euthanized or subject to any variation from the operators ments were found to be generally less effective in limiting licensed processing procedures, for the purposes of the study lactic acid levels and generating acetic and propionic acid. (NHMRC 2013). The handling and preparation of the rumen As such, for the purposes of visual clarity the results for these content was the same as previously described (Luo et al. 2017a). treatments have been omitted from Fig. 1. The introduction of In addition, glucose (1%, by weight) was added to rumen con- S. bovis alone had little effect (P > 0.05) on the acid profile tent samples in all preparations to simulate the effect of a high relative to that observed for the control; however, the levels of concentrate carbohydrate diet in stimulating the growth and lactic acid accumulation were markedly less (P > 0.05) in the lactic acid production of S. bovis. The preparation of the bacteria preparations containing either propionibacteria, M. elsdenii,or followed the same procedure as described previously (Luo et al. both. The acetic and propionic acid concentrations were both Ann Microbiol (2018) 68:153–158 155 Table 1 Sample sets prepared for Group Inoculants analysis of the effects of inoculation of different bacterial Control 2 ml saline combinations on acid metabolism in the rumen content SB 1 ml Streptococcus bovis+1 ml saline Two-strain-inoculation PJ702 + SB 1 ml S.bovis+1 ml Propionibacterium jensenii 702 PA25562 + SB 1 ml S.bovis+1 ml Propionibacterium acidopropionici ATCC 25562 PA341 + SB 1 ml S.bovis+1 ml P. acidopropionici 341 PF2206 + SB 1 ml S.bovis+1 ml P. freudenreichii CSCC 2206 PF2207 + SB 1 ml S.bovis+1 ml P. freudenreichii CSCC 2207 ME+SB 1 ml S.bovis+1 ml Megashaera elsdenii Three-strain-inoculation PJ702 + ME+SB 1 ml S.bovis+1 ml P. jensenii 702 + 1 ml M.elsdenii Inoculate environment for each group listed above was 300 g rumen sample with 1% glucose (w/w) higher in the treatments than in the control preparation. The across all treatments was 26.94 mM. Significant differences results of the three-strain inoculation relative to the two-strain between treatments were evident for the propionic acid con- preparations suggest that strain PJ702 was more influential on centration curves during the incubation (P <0.001) (Fig. 1c). the acid profile than M. elsdenii, and that the effects were not For treatments PJ702, PA341 and PJ702 + ME, the propionic diminished by their co-cultivation. acid concentration rapidly increased to above 30 mM at 24 h, Significant differences were apparent for the lactic acid remaining relatively constant after this time point. Treatments concentration curves between different treatments in the ru- ME, SB and control all exhibited lower propionic acid con- men samples during the incubation period (P < 0.01) (Fig. 1). centration levels across the incubation period. At 24 h, their Lactic acid was not detected in any of the preparations over propionic acid concentrations (11.21 mM, 11.45 mM and the first 4 h of incubation. In all treatments, significant in- 12.40 mM, respectively), less than one-half the levels record- creases in lactic acid concentration were observed with peaks ed for the other preparations. The final concentrations for in concentration observed after either 8 h (for ME, PA 341) or treatment SB and control were 24.98 mM and 22.81 mM re- 12 h (for control, SB, PJ702 and PJ702 + ME). Peak lactic spectively, while further increases were not observed in the ME treatment. acid concentrations for the control and SB treatments (52.60 mM and 55.3 mM, respectively) were significantly The rumen samples used in this study were directly trans- (P < 0.01) higher than those observed for the ME treatment ferred from whole rumen content, which contains a large (35.6 mM) or the propionibacteria treatments (PA341, amount of semi-digested feed (digesta) and indigenous rumen 20.70 mM; PJ702, 21.60 mM; PJ702 + ME, 20.90 mM). micro-flora. The handling of the rumen content was carried out Lactic acid levels returned to below detection limits after carefully under aseptic procedure to preserve the indigenous 24 h in treatments ME, PA341, PJ702 and PJ702 + ME. This microorganisms as much as possible, and avoid the introduction ‘end point’ was not reached in the control and SB treatments, of any contamination to the rumen system, providing a rumen even at the end of the 48 h incubation (Fig. 1a). fermentation environment as close as possible to the normal Marked increases in acetic acid levels were apparent in all in vivo conditions. Interactions between propionibacteria preparations (P = 0.008) (Fig. 1b). The initial average acetic strains, M. elsdenii and S. bovis were subsequently investigated, acid concentration was 12.92 mM across all treatments, steadily and significant differences in acid profiles were observed be- increasing to ~ 45 mM by the end of the incubation. Significant tween treatments and control. The key finding was that, in differences were evident between treatments for the acetic acid comparison with M. elsdenii, dairy propionibacteria exhibited concentration curves (P = 0.04). Treatment PJ 702 recorded the greater capacity to limit the accumulation of lactic acid pro- highest acetic acid level at both 24 h and 48 h with a final duced by S. bovis in rumen content samples, under conditions concentration of 48.99 mM, with similar final concentrations conducive to the development of acidosis. observed for both the PJ702 + ME and SB treatments. The low- In accordance with the negligible levels normally observed est acetic acid levels at both 24 h and 48 h (29.83 mM and in the rumen of healthy animals (Owens et al. 1998;Russell 30.48 mM respectively) were observed in the ME treatment. and Rychlik 2001), lactic acid was not detected in these rumen The initial propionic acid concentration in rumen samples samples prior to inoculation and incubation. The addition of was zero across all preparations, but was detectable after a 2-h excess glucose (1% w/v) clearly appeared to promote the pro- incubation. The average final propionic acid concentration duction and accumulation of lactic acid, indicating successful 156 Ann Microbiol (2018) 68:153–158 PJ702+SB Fig. 1 Change in a lactic, b acetic 60 PA341+SB and c propionic acid ME+SB concentrations in the various treatments in two-strain- SB inoculation rumen cultures. Control Details of each treatment are as PJ 702+ME+SB listed in Table 1.Each point represents the mean value of replicate measurements (n =3) 0 4 8 1216 20242832 36404448 Time (hours) PJ702+SB PA341+SB ME+SB SB Control PJ 702+ME+SB 0 4 8 121620242832 36404448 Time (hours) PJ702+SB PA341+SB ME+SB SB Control PJ 702+ME+SB 0 4 8 12162024283236404448 Time (hours) establishment of simulated ruminal acidosis conditions. The by the control samples, it should be recognized that all rumen extra glucose introduced into the rumen would have stimulat- samples received a fixed supply of glucose, thus limiting the ed the growth of indigenous lactic acid producing bacteria, maximum amount of lactic acid that could be produced. That enabling them to generate large amounts of lactic acid in a is, despite the elevated presence of S. bovis, production of short period of time, as evidenced by the exponential increase lactic acid was ultimately limited by the finite availability of of lactic acid in the control samples to 52.65 mM during the substrate. first 12 h. Such a reaction reflects the progression of lactic The most significant finding was the reduction in lactic acidosis in the rumen, with glucose serving as the easily fer- acid accumulation in rumen cultures inoculated with either mentable carbohydrate. The fact that the SB treatment, con- strains of Propionibacterium, M. elsdenii, or both. The peak taining inoculation of S. bovis only, produced the highest level lactic acid concentrations in those treatments were substantial- of lactic acid production of any of the treatments appeared to ly lower compared with the control and the SB treatment. confirm its role as a major lactic acid producer in the rumen. Moreover, after reaching peak levels, the lactic acid in the While this peak level was only 5% greater than that produced rumen samples was reduced and eventually eliminated in the Concentraon (mM) Concentraon (mM) Concentraon (mM) Ann Microbiol (2018) 68:153–158 157 treatments inoculated with propionibacteria or M. elsdenii are able to utilize lactic acid as a carbon source, and this could (Fig. 1). This result has successfully demonstrated that the explain why the introduction of lactic acid in rumen samples application of dairy propionibacteria, M. elsdenii, or their in the previous study (Luo et al. 2017b) had limited effect on combination in the rumen, was able to prevent the accumula- elevation of acetic acid concentration levels. tion of large amounts of lactic acid and remove them from the In the three-strain-bacterial inoculation, Propionibacterium rumen content samples completely. strain PJ702 appeared to have a stronger influence on acid Similarly, reduction of lactic acid by inoculation of metabolism than M. elsdenii where both existed in the rumen M. elsdenii in a simulated rumen acidosis environment was sample. In terms of lactic acid levels, no synergistic effect was reported by Kung and Hession (1995), where elevation of evident when using two lactate utilisers together. Although the lactic acid to a concentration of 50 mM was observed in the lactic acid concentration was lowest at 8 h in the three strain glucose- and maltose-enriched rumen fluid medium in the first preparation (PJ702 + ME+SB) than in the two-strain treat- 12 h. The inoculation of M. elsdenii was able to prevent the ments, the peak level of lactic acid in treatment PJ702 + accumulation of lactic acid. This finding is in agreement with ME+SB was similar to that in treatment PJ702 + SB, which the results of the present study, where similar levels of lactic appeared in both cases at 12 h. The acetic and propionic acid acid reduction were also observed in the treatments containing concentration was significantly higher in the treatment Propionibacterium spp. It is important to note however, the PJ702 + ME+SB than the ME+SB and comparable to those differences in the composition of the growth environment be- in the PJ702 + SB and PA341 treatments. These results indi- tween the present and previous studies. In the Kung and cate that the majority of the carbon source was converted to Hession (1995) study, the medium comprised filtered rumen acetic and propionic acid during the incubation. This implies fluid with several additives, including cysteine HCL and malt- that the metabolic activity of strain PJ702 was largely ose, to enhance the growth of M. elsdenii. In the present study, overpowering the metabolic activity of M. elsdenii in the ru- the medium was whole rumen content comprising unmodified men sample during the incubation. digesta and indigenous micro-flora, and no extra enhancement It would appear that the characteristic physiological prop- other than the glucose. The same level of lactic acid elevation erties of M. elsdenii may restrict its potential for application in in the control and reduction of lactic acid accumulation in the treating ruminal acidosis. Firstly, M. elsdenii is sensitive to a treatment in both studies confirm that the application of lactic low pH environment, and exposure to pH values lower than 5 acid utilizing bacteria has potential for the treatment and pre- can have a severe impact on the metabolism and survival of vention of ruminal acidosis. this bacterium (Russell et al. 1981). Secondly, M. elsdenii is Among the tested dairy propionibacteria, strains PJ702 and sensitive to oxygen. Van Dijk et al. (1980)reportedthatthe PA341 have shown greater capacity for lactic acid consump- dehydrogenase enzyme isolated from M. elsdenii was highly tion than other strains as well as their lactic aid consumption in sensitive to oxygen, and that partial inactivation occurs even previous studies in SLB medium (Luo et al. 2017b) and rumen before oxygen can be detected in the bacterial broth. samples (Luo et al. 2017a). In the study of Luo et al. (2017a), Therefore, the sensitivity of M. elsdenii to the surrounding no extra S. bovis was introduced to the rumen content samples, environment may severely impair its capacity for lactic acid and the lactic acid in the rumen was provided before the inoc- consumption, which appeared evident in the present study. On ulation of bacteria, while in the present study the lactic acid in the other hand, dairy propionibacteria, seemingly more resil- the rumen samples was produced by the S. bovis, which grew ient bacteria with higher tolerance to low pH and oxygen on the available glucose in the environment. This change in the (dairy propionibacteria are facultative bacteria) and demon- rumen culture environment appeared to provide different im- strated capacity for lactic acid consumption, may be a more pacts on the metabolism of these strains of Propionibacterium. feasible option for application in the treatment and prevention Compared with the previous study (Luo et al. 2017b), the of ruminal acidosis. production of propionic acid had similar profiles between dif- In this study, it was hypothesized that the acid profile ferent preparations. In relation to the acetic acid profile, the would vary significantly based on the inoculation of different control produced a higher final concentration (45.55 mM) in combinations of bacteria, and this was clearly confirmed. The the present study than the previous study (27.96 mM). This most significant finding was the suppression of lactic acid may reflect the addition of extra glucose in the present study accumulation by the dairy propionibacteria such as PJ702 rather than lactic acid in the previous study. Many indigenous and PA341 during the growth of S. bovis in the simulated bacteria in the rumen are able to use glucose to produce acetic rumen environment. Moreover, these dairy propionibacteria and propionic acid. S. bovis itself and other common cellulo- demonstrated superior lactic acid consumption capacity over lytic bacteria in the rumen such as Fibrobacter succinogenes that of M. elsdenii. Although the synergistic effect of applying (Weimer 1993), Ruminococcus flavefaciens (Shi and Weimer both dairy propionibacteria and M. elsdenii together was not 1992)and Ruminococcus albus (Pavlostathis et al. 1988)all shown to be strong, in the actual rumen environment the re- have this capacity. In contrast, very few bacteria in the rumen moval of excessive lactic acid by the application of these 158 Ann Microbiol (2018) 68:153–158 Maroune M, Bartos S (1987) Interactions between rumen amylolytic and propionibacteria may create more favorable conditions for the lactate-utilizing bacteria in growth on starch. J Appl Microbiol 63: recovery of M. elsdenii numbers, thereby helping to restore 233–238 the fermentation process in the rumen. Under such circum- Millet S, Maertens L (2011) The European ban on antibiotic growth stances, the cooperation of suitable strains of propionibacteria promoters in animal feed: from challenges to opportunities. Vet J 187(2):143–144 and M. elsdenii may be beneficial in preventing the occurrence Miwa T, Abe T, Fukuda S, Ohkawara S, Hino T (2000) Effect of reduced of ruminal acidosis more effectively. H+-ATPase activity on acid tolerance in Streptococcus bovis mu- tants. Anaerobe 6:197–203 Compliance with ethical standards NHMRC (2013) Australian code for the care and use of animals for scientific purposes (8th edn). National Health and Medical Conflict of interest The authors declare no conflict of interest. Research Council, Canberra Oshio S, Tahata I, Minato H (1987) Effect of diets differing in ratios of roughage to concentrate on microflora in the rumen of heifers. J Gen Appl Microbiol 33:99–111 References Owens FN, Secrist DS, Hill WJ, Gill DR (1998) Acidosis in cattle: a review. J Anim Sci 76:275–286 Pavlostathis SG, Miller TL, Wolin MJ (1988) Fermentation of insoluble Dieho K, Dijkstra J, Schonewille JT, Bannink A (2016) Changes in ru- cellulose by continuous cultures of Ruminococcus albus.Appl minal volatile fatty acid production and absorption rate during the Environ Microbiol 54:2655–2659 dry period and early lactation as affected by rate of increase of Russell JB, Dombrowski DB (1980) Effect of pH on the efficiency of concentrate allowance. J Dairy Sci 99(7):5370–5384 growth by pure cultures of rumen bacteria in continuous culture. Enemark JMD (2008) The monitoring, prevention and treatment of sub- Appl Environ Microbiol 39(3):604–610 acute ruminal acidosis (SARA): a review. Vet J 176:32–43 Russell JB, Hino T (1985) Regulation of lactate production in Hino T, Shimada K, Maruyama T (1994) Substrate preference in a strain Streptococcus bovis: a spiraling effect that contributes to rumen of Megasphaera elsdenii, a ruminal bacterium, and its implications acidosis. J Dairy Sci 68:1712–1721 in propionate production and growth competition. Appl Environ Russell JB, Rychlik JL (2001) Factors that alter rumen microbial ecology. Microbiol 60:1827–1831 Science 292:1119–1122 Holt JG, Krieg NR, Seneath PHA, Staley JT, Williams ST (1994) Russell JB, Cotta MA, Dombrowski DB (1981) Rumen bacterial compe- Bergey’s manual of determinative bacteriology. Williams and tition in continuous culture: Streptococcus bovis versus Wilkins, Baltimore Megasphaera elsdenii. Appl Environ Microbiol 41:1394–1399 Kung L, Hession AO (1995) Preventing in vitro lactate accumulation in Shi Y, Weimer PJ (1992) Response surface analysis of the effects of pH ruminal fermentations by inoculation with Megasphaera elsdenii.J and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose- Anim Sci 73:250–256 fed continuous culture. Appl Environ Microbiol 58:2583–2591 Luo J, Ranadheera CS, King S, Evans C, Baines S (2017a) In vitro in- Van Dijk C, Grande HJ, Mayhew SG, Veeger C (1980) Properties of vestigation of the effect of dairy propionibacteria on rumen pH, lactic acid and volatile fatty acids. J Integr Agric 16(7):1566–1575 the Hydrogenase of Megasphaera elsdenii. Eur J Biochem 107: Luo J, Ranadheera CS, King S, Evans C, Baines S (2017b) Potential 251–261 influence of dairy propionibacteria on the growth and acid metabo- Weimer PJ (1993) Effects of dilution rate and pH on the ruminal cellulo- lism of Streptococcus bovis and Megasphaera elsdenii. Benefic lytic bacterium Fibrobacter succinogenes S85 in cellulose-fed con- Microbes 8(1):111–119 tinuous culture. Arch Microbiol 160:288–294 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Dairy propionibacteria as direct-fed microbials: in vitro effect on acid metabolism of Streptococcus bovis and Megasphaera elsdenii

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Springer Journals
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Copyright © 2018 by Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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Abstract

Ruminal acidosis caused by accumulation of lactic acid, a decrease of pH in the rumen and subsequent imbalance of the rumen fermentation process, affects the health and productivity of dairy cows and beef cattle. Direct-fed microbials have potential for use in the control and prevention of ruminal acidosis. This study investigated the interaction between five strains of dairy propionibacteria, Megasphaera elsdenii and Streptococcus bovis in various co-culture combinations in a simulated rumen environment comprising unmodified rumen digesta supplemented with excess glucose. While suppression of lactic acid accu- mulation by both the dairy propionibacteria and M. elsdenii in the presence of S. bovis in the simulated rumen conditions was evident, propionibacteria were found to be more effective than M. elsdenii in controlling lactic acid levels. . . . Keywords Probiotics Propionibacteria Rumen acidosis Rumen microbes Findings haboring a variety of microorganisms, some of which have been shown to influence the development of acidosis. Propionibacteria are characterized by utilization of lactic acid Streptococcus bovis is an inhabitant of the rumen environ- as the favored carbon source, with propionic acid produced as ment, but is usually found in relatively low numbers in the a by-product (Luo et al. 2017a). Dairy propionibacteria have healthy rumen. However, S. bovis is relatively acid tolerant been proposed as potential probiotic candidates for the treat- (Russell and Hino 1985; Miwa et al. 2000) and has been ment and prevention of ruminal acidosis—a prevalent disor- identified as the major lactic acid producer responsible for der in ruminants (Luo et al. 2017b). Ruminal acidosis is the development of acidosis (Maroune and Bartos 1987; caused primarily by the inclusion of a high percentage of Owens et al. 1998;Enemark 2008). Although antibiotic treat- readily fermentable dietary carbohydrates. This disorder pre- ment has proven to be effective in treating this condition, sents as an accumulation of lactic acid with a decrease of pH in potential facilitation of the spread of antibiotic-resistant bac- the rumen and subsequent imbalance of the rumen flora and teria makes this an unattractive option for routine use in fermentation processes, resulting in impaired health and pro- preventing acidosis (Millet and Maertens 2011). Therefore, ductivity of dairy cows and feedlot beef cattle (Enemark 2008; alternative approaches such as use of direct-fed microbials Luo et al. 2017a, b). The rumen is a complex environment (DFM) to control the growth of S. bovis are becoming increas- ingly popular (Luo et al. 2017b). Megasphaera elsdenii, the major lactic acid utilizer in the rumen, is able to use lactate, fructose and glucose as carbon * Chaminda Senaka Ranadheera sources, and produces propionate, acetate and butyrate as ma- senakar@email.com jor metabolic products (Holt et al. 1994). In the healthy rumen, M. elsdenii mainly utilizes the maltose hydrolyzed from starch School of Environmental and Life Sciences, The University of and the lactate produced by S.bovis as carbon sources (Russell Newcastle, Callaghan, NSW 2308, Australia et al. 1981;Hino et al. 1994), and, therefore, maintains the School of Agriculture and Food, Faculty of Veterinary and lactic acid level. However, this balance is often disrupted when Agricultural Sciences, The University of Melbourne, Melbourne, VIC 3010, Australia cattle are fed concentrates that include a higher percentage of starch, because it stimulates the growth of amylolytic bacteria School of Health Sciences, The University of Newcastle, to produce more volatile fatty acids (VFAs) and glucose. Since Callaghan, NSW 2308, Australia 154 Ann Microbiol (2018) 68:153–158 S. bovis can use both starch and glucose as carbon sources, this 2017b). Maximum grown (~10 cfu/ml) and saline-washed bac- increasing availability of substrate stimulates the rapid growth terial preparations of propionibacteria, S. bovis and M. elsdenii of S. bovis and leads to accumulation of lactic acid causing were used as inoculants. The five strains of dairy acidification in the rumen (Hino et al. 1994). The effects are propionibacteria used in the study, along with the abbreviated further exacerbated by the vulnerability of M. elsdenii to acidic names in brackets used throughout this paper, were conditions (Russell and Dombrowski 1980), which reduces Propionibacterium jensenii 702 (PJ702), P. acidopropionici their capacity for lactic acid consumption. ATCC 25562 (PA25562), P. acidopropionici 341 (PA341), Although propionibacteria are common inhabitants in the P. freudenreichii CSCC 2206 (PF2206) and P. freudenreichii rumen, they are normally present in low numbers (Oshio et al. CSCC 2207 (PF2207). There were ten preparations assigned 1987) and therefore exert no significant influence in control- in this study, both as two-strain and three-strain co-cultures. ling the development of acidosis. However, it has been dem- The details of the inoculation for each preparation are listed in onstrated that the introduction of dairy propionibacteria to a Table 1. Both two- and three-strain co-culture studies were per- nutrient broth medium containing co-cultures of S. bovis and formed. No additional bacterium was inoculated to the control. M. elsdenii was able to influence the fermentation process as For the treatment SB, only one strain of bacteria (S. bovis)was the lactic acid was efficiently converted into acetic acid and introduced. Two strains of bacteria were inoculated to treat- propionic acid (Luo et al. 2017b). These VFAs produced by ments PJ702 + SB, PA25562 + SB, PA341 + SB, PF2206 + dairy propionibacteria can then be absorbed through the rumen SB, PF2207 + SB and ME+SB, which contain S. bovis and wall and serve as an energy source for cattle (Dieho et al. either one strain of Propionibacterium or M. elsdenii.Three 2016). Although these findings indicated that the dairy different strains of bacteria S. bovis, P. jensenii 702 and propionibacteria may have potential in the alleviation of rumi- M. elsdenii were inoculated together for the treatment nal acidosis, given the complexity and the diversity of healthy PJ702 + ME+SB. All inoculated rumen samples were incubated ruminal micro-flora, the influence of other indigenous micro- in a CO incubator (Thermo Electrone, Thermo Scientific, flora on these interactions in the same growth environment Waltham, MA) with 10% CO under 37 °C for 48 h. At 0 h, must also be considered. In relation to this, the interaction 2 h, 4 h, 8 h, 12 h, 24 h and 48 h post inoculation, 5 ml sample between dairy propionibacteria, S. bovis,and M. elsdenii in was taken for high pressure liquid chromatography (HPLC) the rumen environment has not been well documented. analysis for the lactic, propionic, and acetic acid profiles as Hence, this study was designed to examine the metabolic in- described previously (Luo et al. (2017a) using a HPLC system teractions between dairy propionibacteria, S. bovis and (Hewlett-Packard 1100 DAD, Santa Clara, CA) fitted with a M. elsdenii in unmodified rumen fluid. In this context, the Pyrospher RP-18 (125 mm × 4 mm, 5 μm) column (Hewlett- introduction of glucose and S. bovis to the rumen content Packard). Bacterial cell abundances were not quantified during samples was carried out to create in vitro conditions similar analysis. Comparisons of the difference between the acid con- to those of ruminal acidosis. The hypothesis was that, under centration curves against time in different preparations in rumen the simulated ruminal acidosis conditions, the inoculation of contents were performed using the General Linear Model either propionibacteria, M. elsdenii, or their combination, Repeated Measurement in SPSS (PASW statistic 18), to mea- would prevent the accumulation of lactic acid via consumption sure the difference between the trajectories of each individual and conversion to acetic and propionic acids. Moreover, that acid concentration curve across the whole experimental period. the extent of the effect would vary dependent upon the specific In this study, the acid concentration profiles were altered propionibacteria strains involved. significantly by the introduction of different strains of Whole rumens (beef cattle) were obtained post-mortem from Propionibacterium and M. elsdenii in the glucose-fortified ru- a local abattoir (Kurri Meats, Newcastle, Australia) as part of the men content samples inoculated with S. bovis (Fig. 1). Among waste by-product of normal abattoir operations. In accordance the tested propionibacteria, PA341 and PJ702 were the strains with Australian government guidelines for the use of animals found to be associated with the lowest levels of lactic acid for scientific purposes, the study was exempt from ethical ap- accumulation and highest production of acetic and propionic proval requirements on the basis that no live animals were han- acid. By comparison, the PA25562, PF2206, and PF2207 treat- dled, euthanized or subject to any variation from the operators ments were found to be generally less effective in limiting licensed processing procedures, for the purposes of the study lactic acid levels and generating acetic and propionic acid. (NHMRC 2013). The handling and preparation of the rumen As such, for the purposes of visual clarity the results for these content was the same as previously described (Luo et al. 2017a). treatments have been omitted from Fig. 1. The introduction of In addition, glucose (1%, by weight) was added to rumen con- S. bovis alone had little effect (P > 0.05) on the acid profile tent samples in all preparations to simulate the effect of a high relative to that observed for the control; however, the levels of concentrate carbohydrate diet in stimulating the growth and lactic acid accumulation were markedly less (P > 0.05) in the lactic acid production of S. bovis. The preparation of the bacteria preparations containing either propionibacteria, M. elsdenii,or followed the same procedure as described previously (Luo et al. both. The acetic and propionic acid concentrations were both Ann Microbiol (2018) 68:153–158 155 Table 1 Sample sets prepared for Group Inoculants analysis of the effects of inoculation of different bacterial Control 2 ml saline combinations on acid metabolism in the rumen content SB 1 ml Streptococcus bovis+1 ml saline Two-strain-inoculation PJ702 + SB 1 ml S.bovis+1 ml Propionibacterium jensenii 702 PA25562 + SB 1 ml S.bovis+1 ml Propionibacterium acidopropionici ATCC 25562 PA341 + SB 1 ml S.bovis+1 ml P. acidopropionici 341 PF2206 + SB 1 ml S.bovis+1 ml P. freudenreichii CSCC 2206 PF2207 + SB 1 ml S.bovis+1 ml P. freudenreichii CSCC 2207 ME+SB 1 ml S.bovis+1 ml Megashaera elsdenii Three-strain-inoculation PJ702 + ME+SB 1 ml S.bovis+1 ml P. jensenii 702 + 1 ml M.elsdenii Inoculate environment for each group listed above was 300 g rumen sample with 1% glucose (w/w) higher in the treatments than in the control preparation. The across all treatments was 26.94 mM. Significant differences results of the three-strain inoculation relative to the two-strain between treatments were evident for the propionic acid con- preparations suggest that strain PJ702 was more influential on centration curves during the incubation (P <0.001) (Fig. 1c). the acid profile than M. elsdenii, and that the effects were not For treatments PJ702, PA341 and PJ702 + ME, the propionic diminished by their co-cultivation. acid concentration rapidly increased to above 30 mM at 24 h, Significant differences were apparent for the lactic acid remaining relatively constant after this time point. Treatments concentration curves between different treatments in the ru- ME, SB and control all exhibited lower propionic acid con- men samples during the incubation period (P < 0.01) (Fig. 1). centration levels across the incubation period. At 24 h, their Lactic acid was not detected in any of the preparations over propionic acid concentrations (11.21 mM, 11.45 mM and the first 4 h of incubation. In all treatments, significant in- 12.40 mM, respectively), less than one-half the levels record- creases in lactic acid concentration were observed with peaks ed for the other preparations. The final concentrations for in concentration observed after either 8 h (for ME, PA 341) or treatment SB and control were 24.98 mM and 22.81 mM re- 12 h (for control, SB, PJ702 and PJ702 + ME). Peak lactic spectively, while further increases were not observed in the ME treatment. acid concentrations for the control and SB treatments (52.60 mM and 55.3 mM, respectively) were significantly The rumen samples used in this study were directly trans- (P < 0.01) higher than those observed for the ME treatment ferred from whole rumen content, which contains a large (35.6 mM) or the propionibacteria treatments (PA341, amount of semi-digested feed (digesta) and indigenous rumen 20.70 mM; PJ702, 21.60 mM; PJ702 + ME, 20.90 mM). micro-flora. The handling of the rumen content was carried out Lactic acid levels returned to below detection limits after carefully under aseptic procedure to preserve the indigenous 24 h in treatments ME, PA341, PJ702 and PJ702 + ME. This microorganisms as much as possible, and avoid the introduction ‘end point’ was not reached in the control and SB treatments, of any contamination to the rumen system, providing a rumen even at the end of the 48 h incubation (Fig. 1a). fermentation environment as close as possible to the normal Marked increases in acetic acid levels were apparent in all in vivo conditions. Interactions between propionibacteria preparations (P = 0.008) (Fig. 1b). The initial average acetic strains, M. elsdenii and S. bovis were subsequently investigated, acid concentration was 12.92 mM across all treatments, steadily and significant differences in acid profiles were observed be- increasing to ~ 45 mM by the end of the incubation. Significant tween treatments and control. The key finding was that, in differences were evident between treatments for the acetic acid comparison with M. elsdenii, dairy propionibacteria exhibited concentration curves (P = 0.04). Treatment PJ 702 recorded the greater capacity to limit the accumulation of lactic acid pro- highest acetic acid level at both 24 h and 48 h with a final duced by S. bovis in rumen content samples, under conditions concentration of 48.99 mM, with similar final concentrations conducive to the development of acidosis. observed for both the PJ702 + ME and SB treatments. The low- In accordance with the negligible levels normally observed est acetic acid levels at both 24 h and 48 h (29.83 mM and in the rumen of healthy animals (Owens et al. 1998;Russell 30.48 mM respectively) were observed in the ME treatment. and Rychlik 2001), lactic acid was not detected in these rumen The initial propionic acid concentration in rumen samples samples prior to inoculation and incubation. The addition of was zero across all preparations, but was detectable after a 2-h excess glucose (1% w/v) clearly appeared to promote the pro- incubation. The average final propionic acid concentration duction and accumulation of lactic acid, indicating successful 156 Ann Microbiol (2018) 68:153–158 PJ702+SB Fig. 1 Change in a lactic, b acetic 60 PA341+SB and c propionic acid ME+SB concentrations in the various treatments in two-strain- SB inoculation rumen cultures. Control Details of each treatment are as PJ 702+ME+SB listed in Table 1.Each point represents the mean value of replicate measurements (n =3) 0 4 8 1216 20242832 36404448 Time (hours) PJ702+SB PA341+SB ME+SB SB Control PJ 702+ME+SB 0 4 8 121620242832 36404448 Time (hours) PJ702+SB PA341+SB ME+SB SB Control PJ 702+ME+SB 0 4 8 12162024283236404448 Time (hours) establishment of simulated ruminal acidosis conditions. The by the control samples, it should be recognized that all rumen extra glucose introduced into the rumen would have stimulat- samples received a fixed supply of glucose, thus limiting the ed the growth of indigenous lactic acid producing bacteria, maximum amount of lactic acid that could be produced. That enabling them to generate large amounts of lactic acid in a is, despite the elevated presence of S. bovis, production of short period of time, as evidenced by the exponential increase lactic acid was ultimately limited by the finite availability of of lactic acid in the control samples to 52.65 mM during the substrate. first 12 h. Such a reaction reflects the progression of lactic The most significant finding was the reduction in lactic acidosis in the rumen, with glucose serving as the easily fer- acid accumulation in rumen cultures inoculated with either mentable carbohydrate. The fact that the SB treatment, con- strains of Propionibacterium, M. elsdenii, or both. The peak taining inoculation of S. bovis only, produced the highest level lactic acid concentrations in those treatments were substantial- of lactic acid production of any of the treatments appeared to ly lower compared with the control and the SB treatment. confirm its role as a major lactic acid producer in the rumen. Moreover, after reaching peak levels, the lactic acid in the While this peak level was only 5% greater than that produced rumen samples was reduced and eventually eliminated in the Concentraon (mM) Concentraon (mM) Concentraon (mM) Ann Microbiol (2018) 68:153–158 157 treatments inoculated with propionibacteria or M. elsdenii are able to utilize lactic acid as a carbon source, and this could (Fig. 1). This result has successfully demonstrated that the explain why the introduction of lactic acid in rumen samples application of dairy propionibacteria, M. elsdenii, or their in the previous study (Luo et al. 2017b) had limited effect on combination in the rumen, was able to prevent the accumula- elevation of acetic acid concentration levels. tion of large amounts of lactic acid and remove them from the In the three-strain-bacterial inoculation, Propionibacterium rumen content samples completely. strain PJ702 appeared to have a stronger influence on acid Similarly, reduction of lactic acid by inoculation of metabolism than M. elsdenii where both existed in the rumen M. elsdenii in a simulated rumen acidosis environment was sample. In terms of lactic acid levels, no synergistic effect was reported by Kung and Hession (1995), where elevation of evident when using two lactate utilisers together. Although the lactic acid to a concentration of 50 mM was observed in the lactic acid concentration was lowest at 8 h in the three strain glucose- and maltose-enriched rumen fluid medium in the first preparation (PJ702 + ME+SB) than in the two-strain treat- 12 h. The inoculation of M. elsdenii was able to prevent the ments, the peak level of lactic acid in treatment PJ702 + accumulation of lactic acid. This finding is in agreement with ME+SB was similar to that in treatment PJ702 + SB, which the results of the present study, where similar levels of lactic appeared in both cases at 12 h. The acetic and propionic acid acid reduction were also observed in the treatments containing concentration was significantly higher in the treatment Propionibacterium spp. It is important to note however, the PJ702 + ME+SB than the ME+SB and comparable to those differences in the composition of the growth environment be- in the PJ702 + SB and PA341 treatments. These results indi- tween the present and previous studies. In the Kung and cate that the majority of the carbon source was converted to Hession (1995) study, the medium comprised filtered rumen acetic and propionic acid during the incubation. This implies fluid with several additives, including cysteine HCL and malt- that the metabolic activity of strain PJ702 was largely ose, to enhance the growth of M. elsdenii. In the present study, overpowering the metabolic activity of M. elsdenii in the ru- the medium was whole rumen content comprising unmodified men sample during the incubation. digesta and indigenous micro-flora, and no extra enhancement It would appear that the characteristic physiological prop- other than the glucose. The same level of lactic acid elevation erties of M. elsdenii may restrict its potential for application in in the control and reduction of lactic acid accumulation in the treating ruminal acidosis. Firstly, M. elsdenii is sensitive to a treatment in both studies confirm that the application of lactic low pH environment, and exposure to pH values lower than 5 acid utilizing bacteria has potential for the treatment and pre- can have a severe impact on the metabolism and survival of vention of ruminal acidosis. this bacterium (Russell et al. 1981). Secondly, M. elsdenii is Among the tested dairy propionibacteria, strains PJ702 and sensitive to oxygen. Van Dijk et al. (1980)reportedthatthe PA341 have shown greater capacity for lactic acid consump- dehydrogenase enzyme isolated from M. elsdenii was highly tion than other strains as well as their lactic aid consumption in sensitive to oxygen, and that partial inactivation occurs even previous studies in SLB medium (Luo et al. 2017b) and rumen before oxygen can be detected in the bacterial broth. samples (Luo et al. 2017a). In the study of Luo et al. (2017a), Therefore, the sensitivity of M. elsdenii to the surrounding no extra S. bovis was introduced to the rumen content samples, environment may severely impair its capacity for lactic acid and the lactic acid in the rumen was provided before the inoc- consumption, which appeared evident in the present study. On ulation of bacteria, while in the present study the lactic acid in the other hand, dairy propionibacteria, seemingly more resil- the rumen samples was produced by the S. bovis, which grew ient bacteria with higher tolerance to low pH and oxygen on the available glucose in the environment. This change in the (dairy propionibacteria are facultative bacteria) and demon- rumen culture environment appeared to provide different im- strated capacity for lactic acid consumption, may be a more pacts on the metabolism of these strains of Propionibacterium. feasible option for application in the treatment and prevention Compared with the previous study (Luo et al. 2017b), the of ruminal acidosis. production of propionic acid had similar profiles between dif- In this study, it was hypothesized that the acid profile ferent preparations. In relation to the acetic acid profile, the would vary significantly based on the inoculation of different control produced a higher final concentration (45.55 mM) in combinations of bacteria, and this was clearly confirmed. The the present study than the previous study (27.96 mM). This most significant finding was the suppression of lactic acid may reflect the addition of extra glucose in the present study accumulation by the dairy propionibacteria such as PJ702 rather than lactic acid in the previous study. Many indigenous and PA341 during the growth of S. bovis in the simulated bacteria in the rumen are able to use glucose to produce acetic rumen environment. Moreover, these dairy propionibacteria and propionic acid. S. bovis itself and other common cellulo- demonstrated superior lactic acid consumption capacity over lytic bacteria in the rumen such as Fibrobacter succinogenes that of M. elsdenii. Although the synergistic effect of applying (Weimer 1993), Ruminococcus flavefaciens (Shi and Weimer both dairy propionibacteria and M. elsdenii together was not 1992)and Ruminococcus albus (Pavlostathis et al. 1988)all shown to be strong, in the actual rumen environment the re- have this capacity. In contrast, very few bacteria in the rumen moval of excessive lactic acid by the application of these 158 Ann Microbiol (2018) 68:153–158 Maroune M, Bartos S (1987) Interactions between rumen amylolytic and propionibacteria may create more favorable conditions for the lactate-utilizing bacteria in growth on starch. J Appl Microbiol 63: recovery of M. elsdenii numbers, thereby helping to restore 233–238 the fermentation process in the rumen. Under such circum- Millet S, Maertens L (2011) The European ban on antibiotic growth stances, the cooperation of suitable strains of propionibacteria promoters in animal feed: from challenges to opportunities. Vet J 187(2):143–144 and M. elsdenii may be beneficial in preventing the occurrence Miwa T, Abe T, Fukuda S, Ohkawara S, Hino T (2000) Effect of reduced of ruminal acidosis more effectively. H+-ATPase activity on acid tolerance in Streptococcus bovis mu- tants. 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Journal

Annals of Microbiology – Springer Journals

Published: Jan 25, 2018

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