Effects of AlcH and FlaH on nutrient disappearance and fermentation characteristics
Supplementation of FlaH reduced (P < 0.01) the disappearance of DM, OM, CP and starch as compared to the control and AlcH, which had no difference in the nutrient disappearances (Table 2). The reduction of DM disappearance by FlaH consists with the results obtained in our previous study using batch culture technique. We found that the DM disappearance of barley grain linearly decreased from 76.4, 70.7 to 63.8% as the inclusion rate of FlaH increased from 0, 5 to 10% (unpublished data). The reduction in the disappearances of DM and OM by FlaH was primarily resulted from the decreased of CP and starch. It appeared that the effect of AlcH and FlaH on the nutrient disappearances was not associated with their antioxidant activities measured in the present study. In fact, if we admit that the increased the DPPH scavenging or Fe-chelating activity adversely affected the nutrient disappearance as observed with FlaH, the nutrient disappearance with AlcH would be less than that with FlaH because of the greater DPPH scavenging or Fe-chelating activity of AlcH than FlaH. These results suggest that the hydrolysate with chemically determined antioxidant activity may have different response under rumen fermentation condition. Furthermore, the fermentation under current Rusitec condition would be less in oxidative stress compared with in vivo where the oxygen can frequently enter the rumen with feeds and rumination. We also suggest that the hydrolysates may have more like antimicrobial than antioxidant activity in the fermentation. The different bioactivities of AlcH and FlaH in rumen fermentation would be expected, as the activity of protein hydrolysates depends on the specificity of the protease used, hydrolysis conditions and degree of hydrolysis [13]. The flavourzyme is an endo- and exopeptidase enzyme mixture, and mainly produce small peptides and free amino acids, while the alcalase is an endo-protease and mainly generate small- and medium-sized peptides [13]. Furtherly, about 6% of protein in the total protein input was hydrolysate origin as a result of adding FlaH, the decreased CP disappearance with FlaH could be due to the resistance of hydrolysate to microbial degradation. The resistance is related to the amino acid sequences and structures, especially peptides with Gly-Gly, Pro-X or X-Pro residues at the N-terminus, or with modification of N-terminal amino groups [31, 32]. Thus, it is predicted that FlaH generated by flavourzyme is more resistant to rumen microbial digestion, and possess strong bioactivity in modulating rumen fermentation, including reduce the activity of proteolytic and starch utilization microbes. The disappearance of NDF and ADF were not affected by supplementation of either AlcH or FlaH, suggesting that both protein hydrolysates had limited effect on the activity of fibrolytic microbes.
Table 2
Ingredient and chemical composition of experimental diets
Item | Contents |
Ingredient, % | |
Barley silage1 | 10.0 |
Barley grain2, ground | 87.0 |
Supplement3 | 3.0 |
Chemical composition, % of DM | | | | |
DM | 91.6 |
OM | 95.2 |
CP | 11.8 |
NDF | 20.0 |
ADF | 8.0 |
Starch | 50.3 |
1 Composition (DM basis): 31.8% DM, 94.1% OM, 42.9% NDF, 26.7% ADF, 16.4% starch, and 9.6% CP. |
2 Composition (DM basis): 90.2% DM, 98.4% OM, 14.9% NDF, 4.1% ADF, 55.9% starch and 12.4% CP. |
3 Supplied per kilogram of dietary DM: 565 g barley grain, 100 g canola meal, 250 g calcium carbonate, 25 g molasses, 30 g salt, 20 g urea, 0.66 g vitamin E 500 and 10 g premix. The premix in the supplement contained per kilogram of dietary DM: 15 mg of Cu, 65 of mg Zn, 28 mg of Mn, 0.7 mg of I, 0.2 mg of Co, 0.3 mg of Se, 6,000 IU of vitamin A, 600 IU of vitamin D, and 47 IU of vitamin E. |
The pH in the fermenters remained constant throughout the whole experimental period, with slightly higher (P < 0.01) pH with Ant than other treatments (average, 5.76; Table 3). Production of VFA (mmol/d) and individual VFA molar proportion as well as acetate to propionate ratio were not affected with adding AlcH or FlaH except for lower proportion of butyrate with FlaH than control and AlcH. The similar VFA production is consistent with biologically minor difference in the OM disappearance despite of statistical difference between FlaH and control or AlcH. However, AlcH and FlaH had greater (P < 0.01) NH3-N production than control. The greater NH3-N production with FlaH is not expected because of decreased CP degradability by FlaH. It was probably because they provided certain amount of peptides and free amino acid to fulfil partial nitrogen requirements of ruminal microbes. It was reported that ruminal microbes preferred to use peptides or amino acids as a source of nitrogen or as a source of energy, thus lead to accumulation of NH3-N [33]. We speculate that the supplementation of AlcH and FlaH can promote the deaminative activity of high ammonia producing bacteria. Ammonia is mainly produced by the low activity species, but proliferation of the high ammonia producing bacteria will be a problem if certain diets were fed [31]. It was reported that a variety of ruminal bacteria produced ammonia from protein hydrolysates, with strains of Bacteroides ruminicola, Megasphaera elsdenii, and Selenomonas ruminantium being the most active [34].
Table 3
Effect of brewers’ spent grain (BSG) protein hydrolysates or antibiotics on nutrient disappearances and fermentation characteristics in RUSITEC
Item | Treatments1 | SEM | P value |
Con | AlcH | FlaH | Ant |
Nutrient disappearance, % |
DM | 76.5a | 76.1ab | 75.3b | 72.7c | 0.90 | 0.01 |
OM | 77.7a | 77.6a | 76.5b | 74.1c | 0.87 | 0.01 |
CP | 75.1a | 75.4a | 73.4b | 69.7c | 1.12 | 0.01 |
NDF | 40.3a | 38.7a | 38.9a | 31.8b | 1.32 | 0.01 |
ADF | 29.2a | 26.8a | 27.2a | 21.5b | 1.33 | 0.01 |
Starch | 90.7a | 90.8a | 89.5b | 87.8c | 0.92 | 0.01 |
Fermentation characteristics | | | | | | |
pH | 5.74b | 5.78b | 5.77b | 5.85a | 0.02 | 0.01 |
NH3-N, mmol/d | 3.66b | 4.05a | 4.19a | 2.57c | 0.12 | 0.01 |
Total VFA, mmol/d | 54.80a | 54.51a | 53.63a | 48.88b | 0.86 | 0.01 |
Individual VFA, % of total VFA | | | | | |
Acetate (A) | 30.97a | 31.12a | 31.67a | 29.77b | 0.38 | 0.03 |
Propionate (P) | 39.33b | 39.39b | 39.42b | 42.98a | 0.39 | 0.01 |
Butyrate | 20.37a | 19.98a | 19.20b | 16.76c | 0.45 | 0.01 |
BCVFA2 | 2.21a | 2.19a | 2.19a | 1.56b | 0.03 | 0.01 |
Valerate | 4.25b | 4.33b | 4.78b | 7.12a | 0.41 | 0.01 |
Caproate | 2.64a | 2.77a | 2.80a | 1.57b | 0.17 | 0.01 |
A:P | 0.79a | 0.79a | 0.80a | 0.69b | 0.01 | 0.01 |
a, b, c Least square means within a row with different superscripts differ (P < 0.05). |
1 Con = Control, no antioxidant peptide and no antibiotics; AlcH = Alcalase hydrolysates, 10 mg per gram of TMR (DM basis); FlaH = Flavourzyme hydrolysates, 10 mg per gram of TMR (DM basis); Ant = antibiotics, 0.8 mg monensin + 1 mg tylosin per gram of TMR (DM basis). |
2 Branched-chain volatile fatty acids (isobutyrate + isovalerate). |
The supplementation of antibiotics (P < 0.01) decreased disappearance of DM, OM, CP, NDF, ADF and starch compared with other treatments. Therefore, lower (P < 0.01) production of total VFA and higher (P < 0.01) fermenter pH was observed in Ant treatment. Furthermore, Ant altered the individual VFA proportion as compared to the other treatments, with lower (P < 0.01) molar proportion of acetate, butyrate, BCVFA and caproate, and higher (P < 0.01) molar proportion of propionate and valerate, thus led to lower (P < 0.01) acetate to propionate ratio. Our results confirm the general known mode of action of monensin in the rumen [35]. Additionally, supplementation of monensin and tylosin reduced (P < 0.01) the production of ammonia, which was in alignment with previous reports that ionophores could decrease ammonia production by suppressing high ammonia producing microbial population, as well as the peptidelytic and deaminative activity of the bacteria that grow in the presence of ionophores [35]. Although the FlaH had similar effects on reducing CP and starch disappearance as Ant did, fibre disappearance and fermentation pattern were not altered. It is likely that mode of action in the rumen is different between monensin and FlaH. The addition of FlaH protected protein and starch from rumen fermentation, which can let more protein enter the small intestine for digestion and alleviate risk of rumen acidosis.
Effects of AlcH and FlaH on gas and dissolved gas production
H2 was produced during the oxidizing of reduced electron carries (such as NADH and ferredoxin) by membrane-bound hydrogenases of some rumen microbes, which plays an important role in maintaining the oxidation-reduction homeostasis to guarantee rumen anaerobic fermentation [36]. However, the activity of membrane-bound hydrogenase will be quickly inhibited by high H2 pressure, while the methanogenic Archaea can efficiently use H2 to reduce CO2 to keep a low ruminal H2 pressure, but at the price of producing CH4 [37]. Therefore, either reducing the production of H2 or finding alternative sinks for H2 are theoretically feasible to reduce CH4 emission. It is now clear that ionophore antibiotics, like monensin, can decrease CH4 production via inhibiting growth of H2 producing bacteria, without causing side effects to succinate- and propionate-producing bacteria [35]. In the current study, fermentation liquid and gas were analyzed for measuring dissolved gas (dH2 and dCH4) and gas (H2 and CH4) production, respectively. Production of dissolved gas and total gas (L/d) as well as the production of dH2 were not affected by treatments, whereas the production of dCH4 (% of dGas, mg/d or mg/g digested DM), and CH4 (% of gas) were lower (P < 0.01) with FlaH than with control and AlcH (Table 4). Supplementation of either AlcH or FlaH considerably decreased (P < 0.01) the production of H2, expressed as µg/d or µg/g digested DM. Adding monensin and tylosin consistently decreased (P < 0.05) the production of CH4, H2 and dCH4 compared with other treatments. The reduced production of H2 by adding FlaH (-49%) is of interest, and it consistent with the decrease in dCH4 production and in the proportion of CH4 in total gas. However, the molar proportion of propionate was unchanged by adding FlaH despite of the decreased in production of CH4 and H2. The decrease in OM disappearance with FlaH may be unable to produce sufficient H2 to increase propionate production. These results suggest that the FlaH may especially target at methanogenesis. The decreased production of H2 with AlcH without altering the production of CH4 and propionate is not clear. Anyway, the OM disappearance was not affected by AlcH. The present results confirmed the effect of monensin on reducing CH4 and H2 production and increasing propionate production [35]. Although the FlaH showed reduction of CH4 and H2 as well as the reduction in nutrient disappearance, the magnitude of the reduction by FlaH was much less than by monensin, and in particularly, the FlaH did not increase propionate production and alter fermentation pattern. Therefore, it suggests lower activity of FlaH than monensin and different mode of action between the two additives. Future work also needs to be carried out to determine relationships between their physicochemical and techno-functional properties.
Table 4
Effects of brewers’ spent grain (BSG) protein hydrolysates or antibiotics on gas and dissolved gas production in RUSITEC
Item | Treatments1 | SEM | P value |
Con | AlcH | FlaH | Ant |
Dissolved Gas2 | | | | | | |
Total dGas, L/d | 0.95 | 0.96 | 0.96 | 0.94 | 0.02 | 0.17 |
dCH4, % of dGas | 0.72a | 0.68a | 0.63b | 0.53c | 0.04 | 0.01 |
dCH4, mg/d | 4.42a | 4.21a | 3.86b | 3.17c | 0.23 | 0.01 |
dCH4, mg/g DM digested | 0.59a | 0.58a | 0.53b | 0.45c | 0.03 | 0.01 |
dH2, µg/d | 0.48 | 0.63 | 0.58 | 0.54 | 0.20 | 0.38 |
dH2, µg/g DM digested | 0.06 | 0.08 | 0.08 | 0.07 | 0.01 | 0.39 |
Gas Production3 | | | | | | |
Total Gas, L/d | 1.68 | 1.62 | 1.59 | 1.58 | 0.20 | 0.87 |
CH4, % of Gas | 2.47a | 2.55a | 2.11b | 1.32c | 0.12 | 0.01 |
CH4, mg/d | 27.49a | 27.15a | 24.37a | 14.92b | 5.38 | 0.01 |
CH4, mg/g DM digested | 3.80a | 3.77a | 3.49a | 2.19b | 0.72 | 0.01 |
H2, µg/d | 105.1a | 65.3b | 54.3b | 18.0c | 11.01 | 0.01 |
H2, µg/g DM digested | 14.6a | 9.0b | 7.6b | 2.6c | 1.58 | 0.01 |
a, b, c Least square means within a row with different superscripts differ (P < 0.05); |
1 Con = Control, no antioxidant peptide and no antibiotics; AlcH = Alcalase hydrolysates, 10 mg per gram of TMR (DM basis); FlaH = Flavourzyme hydrolysates, 10 mg per gram of TMR (DM basis); Ant = antibiotics, 0.8 mg monensin + 1 mg tylosin per gram of TMR (DM basis); |
2 Samples from d 14 to 15; |
3 Samples from d 9 to 13. |
Effects of AlcH and FlaH on microbial protein synthesis and microbial community
Production of total microbial N (LAB + FPB + FPA) and LAB was less (P < 0.05) with FlaH than control and AlcH, whereas the production of FPA was greater (P < 0.01) with AlcH and FlaH without difference in FPB among treatments (Table 5). Supplementation of monensin and tylosin produced greater (P < 0.01) FPA but less LAB and total microbial N compared to control and AlcH without differing with FlaH except for LAB, which was less (P < 0.01) with Ant than FlaH. Microbial N production efficiency did not differ among treatments. Although the total microbial N production reduced with adding FlaH or Ant, proportion of attached microbial biomass (FPA + FPB) in the total biomass was greater for FlaH (30.1%) or Ant (34.4%) than control (24.4%). Our results suggested that adding FlaH or Ant benefits to microbial colonization to feed particle. As the supplementation of AlcH and FlaH to basal diet increased the microbial population in the FPA fraction, which was interesting because the microbial attachment is essential to the feed digestion. Thus, the FPA samples were selected for high-throughput sequencing to assess the microbial community. Neither numbers of OTUs (Fig. 1A) nor Shannon diversity indices (Fig. 1B) of FPA microbial community were affected by supplementing AlcH and FlaH. Whereas, both indices were reduced (P < 0.05) by Ant. Similarly, the results of NMDS analysis indicated that there was no clustering of the FPA microbial community in control, FlaH and AlcH treatments, but the Ant treatment had more clustered community structure (Fig. 2). The study on the effects of BSG protein hydrolysates on ruminal microbiome is lacking. A study applied a blend of plant essential oils with antioxidant activity demonstrated no effect on ruminal microbiome [38]. Meanwhile, our results that monensin greatly reduced the diversity of FPA microbial community were in agreement with in vitro [39] or in vivo [38, 40] studies.
Table 5
Effect of brewers’ spent grain (BSG) protein hydrolysates or antibiotics on microbial N synthesis and microbial community of FPA in RUSITEC
Item | Treatments1 | SEM | P value |
Con | AlcH | FlaH | Ant |
Production of microbial N2, mg/d | | | | | | |
LAB3 | 60.7a | 60.7a | 53.3b | 48.2c | 1.94 | 0.05 |
FPB4 | 7.0 | 6.2 | 6.4 | 7.0 | 1.04 | 0.51 |
FPA5 | 13.7c | 16.6b | 17.6ab | 19.4a | 1.82 | 0.01 |
Total | 80.3a | 82.5a | 76.2b | 73.5b | 1.83 | 0.05 |
Efficiency of microbial protein6 | 7.0 | 7.1 | 6.9 | 6.9 | 0.26 | 0.45 |
a, b, c Least square means within a row with different superscripts differ (P < 0.05). |
1 Con = Control, no antioxidant peptide and no antibiotics; AlcH = Alcalase hydrolysates, 10 mg per gram of TMR (DM basis); FlaH = Flavourzyme hydrolysates, 10 mg per gram of TMR (DM basis); Ant = antibiotics, 0.8 mg monensin + 1 mg tylosin per gram of TMR (DM basis). |
2 Samples from d 14 to 15. |
3 Liquid associate bacteria. |
4 Feed particle-bound bacteria. |
5 Feed particle-associated bacteria. |
6 Efficiency of microbial protein, mg microbial N production/g OM fermented |
Analysis of the taxonomic composition revealed that AlcH and FlaH had no affect on the relative abundance (RA) of bacteria at phylum level (Fig. 3A). Whereas, the addition of antibiotics reduced (P < 0.05) the RA of Firmicutes and Bacteroidetes, and enhanced (P < 0.05) the RA of Proteobacteria at phylum level. For all treatments, phyla of Firmicutes, Bacteroidetes and Proteobacteria were the most predominant phyla, accounting for approximately 90% of the microbial biomass in FPA, which was consistent with the sequencing results of liquid associated microbial community of in vitro [38] and in vivo studies [39, 40]. This suggested that phyla of Firmicutes, Bacteroidetes and Proteobacteria were predominant in both liquid and particle associated ruminal microbiome. While at the genus level, Prevotella, Succinivibrio, Selenomonas, Shuttleworthia, Schwartzia, Bifidobacterium, Lactobacillus, Dialister and Anaerobiospirillum were among the 10 most abundant genera in FPA microbial community (Fig. 3B) and should be considered as the “core bacteria” associated with feed particle. The FlaH enhanced (P < 0.05) the RA of Prevotella, but reduced the RA of Selenomonas, Shuttleworthia, Bifidobacterium and Dialister as compared to the control. The AlcH also had less (P < 0.05) RA of Schwartzia and Bifidobacterium than control. Notably, Ant reduced (P < 0.05) the RA of Prevotella, Selenomonas, Shuttleworthia, Bifidobacterium, Lactobacillus and Dialister, and increased RA of Succinivibrio compared with control. Further Log2 fold change analysis found that several genera from phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria had 5% more change (enhanced or reduced) than control (Fig. 4). Furthermore, genera of Shuttleworthia, Selenomonas, Lactobacillus and Schwartzia from phyla Firmicutes were especially susceptible to FlaH, AlcH and Ant supplementation. These results indicated that FlaH and AlcH had less antibacterial effects as compared to Ant. As F. succinogenes, R. albus, R. flavefaciens, P. ruminicola, E. cellulosolvens, and E. ruminantium are major rumen fibrolytic bacteria [41], the present results indicated that application of BSG protein hydrolysates had no obvious detrimental effect on fibrolytic bacteria. This was supported by no treatment effect of FlaH and AlcH on the disappearances of NDF and ADF.
More susceptible to FlaH and AlcH for genera of Shuttleworthia, Selenomonas, Lactobacillus and Schwartzia from phyla Firmicutes and genera of Bifidobacterium from phyla Actinobacteria, suggested that the BSG protein hydrolysates with antioxidant activity were more potent to Gram-positive bacteria than to Gram-negative bacteria. The underlined mechanism is unknown, but it seemed that this difference might be attributed to the lack of the protective outer membrane in Gram-positive bacteria. As essential oils with antioxidant activity, had a stronger antibacterial activity than other essential oils [42], we speculated that the antibacterial effects of BSG protein hydrolysates might be due to their antioxidant activities. The antioxidant activity of protein hydrolysates relies on the enzymes and methods used during BSG protein hydrolysis. Similarly, differences in effectiveness of monensin on ruminal Gram-positive and Gram-negative bacteria were also observed in the current study, and confirm the monensin sensitivity to Gram-positive bacteria [38].
Rumen microbial community was usually dominated by Prevotella at genus level when fed high grain diets [43]. Members of Prevotella are able to utilise various nutrients such as starch, proteins and non-cellulosic polysaccharides [44, 45], and to convert lactate into propionate to prevent accumulation of lactate [40]. Moreover, as Prevotella are also H2-consuming bacteria in addition to Selenomonas [46], the reduced H2 with FlaH and AlcH was likely due to the increased relative abundance of genus Prevotella. Meanwhile, members of Prevotella are known as the predominant proteolytic bacteria with a great diversity of extracellular proteolytic activities in the rumen [31, 47], thus, the decreased disappearance of CP by supplementing monensin could be explained by decreased relative abundance of Prevotella. Currently, the probiotics such as direct-fed microbials are often developed from the genera Lactobacillus and Bifidobacterium [48], and the reduced relative abundance of Lactobacillus and Bifidobacterium with Ant suggested a disadvantage of applying monensin in the diet. The genus Schwartzia are asaccharolytic and can ferment succinic acid to produce propionic acid [49]. It was reported that bacteria affiliated with Schwartzia were negatively correlated with methane emissions [50], which was contrary to our results that monensin reduced the RA of Schwartzia, as well as methane emission.
There is limited information on the genus Shuttleworthia, and its function in the rumen is almost unknown [43]. Recent studies found that genus Shuttleworthia was digesta-adherent rumen bacteria in dairy and beef cattle [51], and was a starch and sugar fermenter [52]. It was reported that supplementation of phytogenic compounds decreased the RA of Shuttleworthia in dry cow [52]. In the current study, the decreased Shuttleworthia by BSG hydrolysates or Ant may explain the decreased starch digestibility with FlaH and Ant. A recent study showed negative correlation of Shuttleworthia with lactate and NH3-N concentration [53], thus explained the greater NH3-N production with BSG hydrolysates.
Bacteria from genus Selenomonas, besides of degrading starch and cellulose, play critical role in maintaining normal rumen fermentation through converting lactate and succinate into propionate and reducing lactate accumulation [54–56] and maintaining low H2 concentration as H2-consumer [57, 58]. As fumarate and nitrate reducers, Selenomonas were proven a significant H2 sinks in sheep [59]. Ruminants fed high starch diets often result in great rumen dH2 concentration, and would promote the growth of S. ruminantium for disposing electrons derived from fermentation [57]. Therefore, in the present study, the reduced H2 production by applying BSG protein hydrolysates or monensin is consistent with the declined relative abundance of Selenomonas.
Members of genus Succinivibrio can ferment both starch and cellulose into succinate, and succinate then fermented by Selenomonas and other bacteria into propionate via succinate pathway, which is the primary propionate producing pathway in rumen [56]. Therefore, the abundances of members of Succinivibrio in the rumen has been reported to be positively associated with feed efficiency of ruminants [60, 61]. The increased RA of Succinivibrio with monensin in the present study, was correlated with the increased propionate proportion and the increased fermenter pH. Our results agreed with previous report that monensin can lead to an increase in abundance of succinate producers [38], and the report that Succinivibrio is positively correlated to the ruminal pH [55, 62]. The increased pH with Ant would be partly due to the increased RA of genus Dialister, which were reported to play a role in altering the buffering capacity of rumen fluid [63]. Furtherly, although the effects of monensin on methanogens were not determined, the application of monensin reduced the H2 production and CH4 emission. Our results supported that the decreased methane production with Ant most likely ascribed to decrease in nutrient availability for methanogenesis by acting on other rumen microorganisms instead of a direct effect of monensin on the methanogens [38].