Evaluation of iso-α-acid and β-acid extracts from hops (Humulus lupulus L.) on fermentation by rumen microbes in dual-flow continuous culture fermenters

Animal Feed Science and Technology 260 (2020) 114385

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Evaluation of iso-α-acid and β-acid extracts from hops (Humulus lupulus L.) on fermentation by rumen microbes in dual-flow continuous culture fermenters

T

Isaac J. Salfer1, Samuel W. Fessenden2, Marshall D. Stern* Department of Animal Science, University of Minnesota, Saint Paul, 55108, United States

A R T IC LE I N F O

ABS TRA CT

Keywords: Rumen Continuous culture Hops Iso-alpha acids Beta-acids

Hops (Humulus lupulus L.), primarily used in the brewing industry, have been shown to have bacteriostatic properties against Gram-positive bacteria. Active antimicrobial compounds contained within hop flowers include iso-α-acids (isohumulones) and β-acids (lupulones). Previous experiments have demonstrated effects of hop extracts on rumen fermentation in batch culture. The objective of this study was to determine the direct effects of iso-α-acids and β-acids on rumen fermentation in continuous culture fermenters. Two experiments were conducted using dual-flow continuous culture fermenters. Each experiment utilized eight fermenters in two consecutive 10 d periods. Within both experiments, the same basal diet consisting of 44 % corn silage, 14 % alfalfa hay, 13 % ground corn, 11 % protein mix, 10 % corn gluten feed, 5 % cottonseed and 3 % liquid vitamin and mineral supplements on a DM basis was provided to the fermenters at a rate of 75 g of DM/L of fermenter volume/day. In experiment 1, hop beta-extract was added daily to the artificial saliva buffer to supply 0 (CON), 600 (LOW), 1200 (MED), or 1800 (HIGH) mg of βacids/kg of diet DM/day. In experiment 2, hop iso-α extract was provided to fermenters via the artificial saliva buffer to supply 0 (CON), 600 (LOW), 1200 (MED), or 1800 (HIGH) mg of iso-αacids/kg of diet DM/day. Data in both experiments were analyzed as a randomized complete block design with experimental period serving as a block and all treatments equally represented within each block. Data were statistically analyzed using GLM procedure of SAS with a model including the fixed effects of experimental period (block), treatment, and the interaction of treatment and period. In experiment 1, mean and maximum fermentation pH increased (P = 0.005) linearly with increasing levels of beta extract inclusion. Additionally, time spent below pH 5.8 increased and time between pH 5.8 and 6.2 decreased (P = 0.007) linearly with greater beta extract inclusion (P = 0.08). However, beta extract did not affect DM, OM, NDF or ADF digestion. Furthermore, beta extract did not modify VFA production or N metabolism. In experiment 2, iso-alpha extract also increased average pH, and tended to increase time above pH 6.2. Iso-α extract did not affect DM, OM, NDF and ADF digestion, VFA production, or N metabolism. Results indicate that increasing the concentration of beta extract or iso-α extract increase rumen pH within continuous culture, but neither extract affects rumen nutrient digestion.

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Corresponding author at: Department of Animal Science, University of Minnesota, 1364 Eckles Ave, Saint Paul, 55108, United States. E-mail address: [email protected] (M.D. Stern). 1 Current affiliation: South Dakota State University Dairy and Food Science Department, Brookings, SD 57006. 2 Current affiliation: Agricultural Modeling and Training Systems (AMTS), LLC, Groton, NY 13073. https://doi.org/10.1016/j.anifeedsci.2019.114385 Received 22 July 2019; Received in revised form 10 December 2019; Accepted 21 December 2019 0377-8401/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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1. Introduction Ionophore antibiotics are commonly included in ruminant diets to improve feed efficiency and reduce excess production of ammonia (NH3) and methane (CH4) from the rumen (Bergen and Bates, 1983). However, public concerns regarding the use of antibiotic growth promotants has created a need to search for alternative rumen modifiers (García-González et al., 2008). Compounds receiving attention as possible ionophore-like alternatives include extracts from hops (Humulus lupulus L.), which have shown potential to reduce ruminal ammonia production (Flythe, 2009). Hops have been predominantly used as a preservative and flavor enhancer in beer production and contain several compounds that exhibit bacteriostatic properties against Gram-positive bacteria and protozoa including iso-α-acids (isohumulones), β-acids (lupulones) and xanthohumol (Haas and Barsoumian, 1994; Srinivasan et al., 2004). These compounds dissipate the transmembrane pH gradient, destroying the proton motive force across cells in a similar matter to ionophores (Sakamoto and Konings, 2003). Previous studies using whole or spent hops show some beneficial changes to fermentation but results are often variable and are confounded by the presence of additional fermentable matter and other antimicrobial compounds such as condensed tannins (Krishna et al., 1986; Narvaez et al., 2011; Wang et al., 2010). Extraction of iso-α and β-acids from hop resin using supercritical CO2 allows for more targeted research using these compounds. Hop extracts decreased hyper ammonia-producing bacteria (HAB) in pure culture (Flythe, 2009), and decreased CH4, ammonia nitrogen and A:P ratio when hops extracts were administered in mixed batch culture (Narvaez et al., 2013a). Alternately, Drouillard et al. (2009) found no effects of hops on total bacteria, S. bovis, methanogens, VFA profiles or ruminal lactate when steers were fed 30 mg β-acids/kg of diet DM. Storlien et al. (2012) observed modest decreases in A:P ratio and CH4 production in rumen batch cultures incubated with hops extract, but the changes were unlikely to be biologically relevant. More recently, Flythe et al. (2017) detected reductions in the HAB species Acetoanaerobium sticklandii strain SR (formerly Clostridium sticklandii SR), and a reduction in ammonia production in mixed culture after supplementation with hop extracts containing 44.64 % α-acids and 24.28 % β-acids. Further research using alternative models may allow a better understanding of the effects of hop extracts on rumen fermentation. Studying iso-α- and β-acid extracts in a continuous culture rumen fermentation system will allow for targeting examination of these effects in a simulated rumen environment. Additionally, to our knowledge, there have been no experiments examining the direct role of iso-α-acids on rumen fermentation. Two experiments were conducted with the objectives of understanding the effects of increasing doses of either β-acid or iso-α- extracts of hops on rumen fermentation in a continuous culture fermenter system. We hypothesize that increasing the concentration of either β-acids or iso-α-acids will modify rumen fermentation by reducing ammonia-N production and decreasing A:P ratio. By directly applying hop extract acids, and by minimizing aerobic exposure, we expect to more accurately distinguish direct effects of hop acids from other confounding factors. 2. Materials and methods 2.1. Cows and experimental diet Two experiments were performed to test effects of increasing doses of hop β-acids or iso-α-acids on rumen fermentation. In both experiments, continuous culture fermenters were inoculated using mixed rumen fluid from 2 lactating dairy cows at University of Minnesota Dairy Cattle Teaching and Research Center. The University of Minnesota Institutional Animal Care and Use committee approved use of all animals in the experiment (FASS, 2010). Cows were fed the same basal diet formulated to meet or exceed requirements of a lactating Holstein cow producing 40 kg of milk/day with 2.8 % fat and 3.7 % protein (NRC, 2001). The diet consisted of 44 % corn silage, 14 % alfalfa hay, 13 % ground corn, 11 % protein mix, 10 % corn gluten feed, 5 % cottonseed and 3 % liquid vitamin and mineral supplement on a DM basis (Table 1). The experimental diet was the same diet fed to the donor cows. After mixing, a portion of the diet was dried at 60 °C in a forced air oven for 48 h and ground in a Wiley No. 4 laboratory mill (Arthur H. Thomas Co., Philadelphia, PA) to pass through a 2 mm screen. The ground diet was pelleted with a CL-5 California pellet mill (California Pellet Mill Co., Crawfordsville, IN) to a final dimension of 6 mm diameter x 12 mm long. The pelleted diet was placed in shallow trays to air dry for 96 h before storing in plastic containers. Dry matter of pellets was measured periodically during both experiments. 2.2. Experimental design and treatments 2.2.1. Experiment 1: β-extract inclusion Eight continuous culture fermenters (1040 ± 20 ml per fermenter) were used in a completely randomized block design during two consecutive 10 d periods. A commercial Hop β-acid extract (BE) was provided as a 45 % ( ± 1 %) solution with propylene glycol (PG), as determined with high-pressure liquid chromatography (HPLC) by the supplier (S.S. Steiner, Inc., New York, NY). Beta-extract was added daily to the artificial saliva buffer to supply 0 (CON), 600 (LOW), 1200 (MED), and 1800 (HIGH) mg of β-acids/kg of diet DM/d. Concentrations were chosen based on previous data that used hops in rumen cultures (Narvaez et al., 2011). Treatments were randomly assigned within experimental period (block), and replicated within block, resulting in 4 observations per treatment. Sample size was based on > 80 % power of observing a P < 0.05 difference based a standard deviation of 1.2 for dry matter digestion observed in previous experiments performed using this same continuous culture system (Lenth, 2007). To control for differences in PG supply from the beta-extract solution, additional PG was introduced to the CON, LOW, and MED treatments to supply a total of 2200 mg PG/kg of diet DM/day for each treatment, equal to the amount of PG introduced via the HIGH treatment. Additional water 2

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Table 1 Ingredient and chemical composition of basal diet for both experiment 1 and experiment 2. Item

Compositiona

Ingredient composition Corn silage Alfalfa hay Ground corn Protein mixb Corn gluten feed Whole cottonseed Liquid vitamin and mineral supplementc

44.2 13.5 12.6 11.0 9.6 5.6 3.4

Chemical composition Crude Protein Soluble nitrogen (% of total N) NDIN (% of total N) ADIN (% of total N) NDF ADF Lignin Ash Starch Sugar NFC Crude fat TDN NEL, 3X (Mcal/kg DM)

17.5 38.5 11.7 5.3 31.5 17.1 3.2 7.4 26.4 5.4 41.3 4.4 73.5 1.73

a

Composition as % of DM unless otherwise noted. Protein mix composition (DM basis): canola meal, 28 %; soybean meal, 22 %; treated soybean meal, 15 %; distillers dried grains, 13 %; ground corn grain, 5 %; calcium carbonate, 5 %; bloodmeal, 3.5 %; sodium bicarbonate, 3.5 %; potassium carbonate, 3 %; trace minerals, 2 %. c Supplement composition (DM basis): Ca, 49 g/kg; P, 11.7 g/kg; NaCl 101 g/ kg; K, 34 g/kg; Mg, 7.2 g/kg; S, 5.5 g/kg; Mn, 1237 mg/kg; Cu, 382 mg/kg; Se, 8.6 mg/kg; Zn, 1813 mg/kg; Vitamin A, 171 IU/kg; Vitamin D, 34 IU/kg; Vitamin E, 706 IU/kg. b

was added to the LOW, MED and HIGH treatments account for the difference in volume due to the addition of PG. Artificial saliva buffer (pH = 8.25) containing the treatments was mixed fresh daily and stored in the dark throughout the experimental periods to prevent light oxidation of β-acids. 2.2.2. Experiment 2: Iso-α-extract inclusion Eight continuous culture fermenters (1045 ± 30 mL) were used in a completely randomized block design during two consecutive 10 d periods. Commercial hop iso-α extract was provided as a 30 % (+/- 2 %) aqueous solution as determined using HPLC by the supplier (S.S. Steiner, Inc., New York, NY). Iso-α extract (IE) was provided to the fermenters through the artificial saliva buffer to supply 0 (CON), 600 (LOW), 1200 (MED) and 1800 (HIGH) mg of iso-α-acids/kg of diet DM/day. These concentrations were chosen to match the concentrations of β-acids added in experiment 1. Treatments were randomly assigned within each experimental period and duplicated within periods to generate 4 experimental units per treatment. Sample size was based on > 80 % power of observing a P < 0.05 difference based a standard deviation of 1.2 for dry matter digestion observed in previous experiments performed using this same continuous culture system (Lenth, 2007). Artificial saliva buffer (pH = 8.10) containing the treatments was mixed fresh daily. Distilled water was added to the saliva buffer to account for differences in volume due to the addition of treatment. Iso-α extract stock solutions and artificial saliva solutions with IE were stored in the dark throughout the experiment to prevent degradation of the iso-αacids. 2.3. Continuous culture operation and collection of rumen fluid inoculum Both experiments used dual-flow continuous culture fermenters, described by Hannah et al. (1986), with a modified pH measurement and control system. Pelleted feed was provided to the fermenters at a rate of 75 g DM/L of fermenter volume/d. An automated feeding system was used to deliver feed in 8 separate 90-minute intervals throughout the day. Artificial saliva buffer was prepared according to Weller and Pilgrim (1974) to provide a final concentration (g/L) of NaHCO3, 5.0; Na2HPO4, 1.76; KHCO3, 1.6; KCL, 0.6; MgSO4, 0.05; and urea, 0.4. During preparation of buffer, nitrogen gas was used to displace oxygen and maintain anaerobiosis. Liquid dilution rate for each fermenter was set to 10 %/h by regulating the artificial saliva input while solids dilution rate was set at 5.5 %/h by regulating liquid output through filters. Individual fermenter pH was measured continuously by an electronic 3

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data acquisition system (DASYLab v5.04, Measurement Computing, Norton, MA) and recorded every 5 min. Fermenter pH was maintained between 5.6 and 6.4 by automated addition of 5 N NaOH or 3 N HCL. Anaerobiosis was maintained within fermenters by addition of N2 gas at a rate of 20 mL/min. Fermenter temperature was maintained at 38.5 ± 0.1 °C. Fermenter contents were mixed using a magnetic stir plate that rotated at a speed of 350 rpm. Rumen fluid inoculum was collected from two ruminally cannulated lactating dairy cows. Composition of the diet fed to the donor cows was formulated to be the same as the experimental diet (Table 1). Rumen contents collected from each cow were collected and transported to the laboratory in pre-warmed thermoses. Contents from each cow were combined in equal parts and strained through 4 layers of cheesecloth. Strained rumen contents were homogenized and divided equally into 8 pre-warmed fermenters. Twenty-five grams of pelleted diet were added to fermenters immediately after inoculation. 2.4. Sample collection Samples were collected on the final 3 d of each 10-d period. Solids and liquid effluents were collected in separate vessels and maintained at 1 °C in a water bath to reduce enzymatic and microbial activity. On sampling days, solids and liquid effluent were combined within fermenter and homogenized using a PT10/3S homogenizer (Kinematica GmbH, Bohemia, NY). Five hundred mL of combined solid and liquid effluent were collected daily from each fermenter during each of the 3 sampling days and composited by fermenter within period so that each sample contained effluent representing 3 d of collection in each period. A portion of the effluent sample was lyophilized and analyzed for DM, OM, NDF, ADF, ash and purines. The remainder of the effluent was frozen and subsequently thawed for analysis of VFA, N and NH3-N. At the end of each 10 d experimental period, fermenter contents were filtered through 4 layers of cheesecloth and centrifuged at 1000 x g to remove feed particles. Supernatant was then centrifuged at 20,000 x g to isolate microbial cells which were collected, lyophilized, and analyzed for DM, OM, total N and purines. 2.5. Chemical analysis Dry matter and ash content of the lyophilized effluent, lyophilized microbial cells and the experimental diet were determined by drying in a forced-air oven at 105 °C for 24 h followed by combustion in a muffle furnace at 550 °C for 24 h (AOAC, 2005). Sequential detergent fiber analysis (Van Soest et al., 1991) was conducted to determine NDF and ADF concentrations of diets and effluents using an ANKOM A200 fiber analyzer with F58 bags (ANKOM Corp, Fairport, NY). Ammonia-N was determined on the supernatant of centrifuged (5000 x g) effluent by steam distillation with magnesium oxide using a Kjeltec 2300 Analyzer (Foss Tecator AB, Höganäs, Sweden). Total N of the diet, effluent, and microbial cells were determined via the Kjeldahl method (AOAC, 1990) using a Kjeltec 2300 Analyzer. Purine concentration of effluent and microbial pellets were determined using a colorimetric assay (Zinn and Owens, 1986). The purine to N ratio (bacterial purine concentration x 0.1573) was used to calculate the flow of bacterial N (g effluent total N x % bacterial N in effluent total N) and OM (g effluent bacterial N/g OM truly digested). Volatile fatty acids were extracted from effluent and measured using an HP6890 gas chromatographer (Hewlett-Packard, Palo Alto, CA) with a 2 m × 6.35 mm × 2 mm Carbopack glass column (Supelco, Bellefonte, PA) as described by (Fessenden et al., 2017). 2.6. Statistical analysis All data processing and analyses were conducted using SAS software version 9.2 (SAS Institute, Inc., Cary, NC, USA). Data in both experiments were analyzed as a randomized complete block design with experimental period serving as a block and all treatments equally represented within each block. Fermenter pH was recorded automatically every 5 min during the 3 d sampling period and was summarized to determine simple mean, minimum and maximum. Time spent below pH 5.8, between pH 5.8 and 6.2 and above 6.2 were calculated using trapezoidal integration. Minutes were calculated from the raw dataset containing readings every 5 min. All results analyzed using the GLM procedure of SAS with the following linear additive model:

Yij = μ+ Pi + Aj + (PA)ij + eij Where μ is the overall mean, Pi is the experimental period (block), Aj is the effect of treatment and (PA)ij is the interaction between period and acid and eij is the error term. For all analyses, differences were considered significant at P ≤ 0.05, with a trend being described at 0.05 < P ≤ 0.10. Differences between treatments were tested using LSMEANS with the PDIFF option. Differences in treatments were reported as orthogonal contrasts to determine linear and quadratic responses to inclusion of either BE or IE. Dunnett’s test was performed to compare the controls in each experiment to each inclusion level of BE or IE, however in both experiments, results were not presented due to non-significance. 3. Results 3.1. Experiment 1: β-extract inclusion Increasing BE concentration increased (P = 0.0007) average fermentation pH (Table 2) but did not affect (P > 0.10) minimum or maximum pH. Furthermore, BE decreased (P = 0.005) time below pH 5.8 and increased (P = 0.007) time between pH 5.8 and 6.2. Time above pH 6.2 tended to be increased (P = 0.08) by increasing IE concentration. While pH was altered by BE inclusion, total VFA concentration was not affected (P > 0.10; Table 3). Similarly, no changes (P > 0.10) in relative concentrations of individual VFA or 4

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Table 2 Effects of increasing concentrations of hop β-extract or iso-α-extract on fermentation pH in continuous culture. Treatmenta

pH

Experiment 1 (β-extract inclusion) Meanc Minimumc Maximumc Time below 5.8, min/d Time between 5.8 and 6.2, min/d Time above 6.2, min/d Experiment 2 (Iso-α-extract inclusion) Meanc Minimumc Maximumc Time below 5.8, min/d Time between 5.8 and 6.2, min/d Time above 6.2, min/d a b c

SEMb

CON

LOW

MED

HIGH

5.73 5.60 6.04 1200 240 0.0

5.76 5.61 6.04 1018 423 0.0

5.79 5.60 6.14 876 562 10.8

5.80 5.61 6.14 806 608 25.8

5.86 5.63 6.18 607 798 34.6

5.83 5.61 6.14 827 425 225

5.92 5.63 6.28 453 843 144

5.94 5.64 6.26 456 763 221

P-Value Linear

Quadratic

0.02 0.01 0.06 100 98 10.8

0.007 0.11 0.12 0.005 0.007 0.08

0.58 0.60 0.92 0.58 0.49 0.48

0.04 0.01 0.06 114 98.2 57.9

0.04 0.27 0.19 0.11 0.48 0.07

0.55 0.31 0.81 0.34 0.14 0.10

CON: 0 mg β-extract or iso-α-extract/kg diet DM; Low: 600 mg/kg diet DM; MED: 1200 mg/kg diet DM; HIGH: 1800 mg /kg diet DM. Standard error of the mean, n = 4 replicates per treatment. Analyzed as repeated measures with 1 observation/fermenter/5 min during 3 consecutive sampling days.

Table 3 Effects of increasing concentrations of hop β-extract or iso-α-extract on VFA concentration and profile in continuous culture. Volatile fatty acids

Experiment 1 (β-extract inclusion) Total VFA, mM VFA, mol/100 mol Acetate Propionate Butyrate Valerate Isobutyrate Isovalerate Branched-chain, mM A:P Ratio Experiment 2 (Iso-α-extract inclusion) Total VFA, mM VFA, mol/100 mol Acetate Propionate Butyrate Valerate Isobutyrate Isovalerate Branched-chain, mM A:P Ratio a b

Treatmenta

SEMb

CON

LOW

MED

HIGH

116.0

119.8

116.9

122.2

51.2 29.1 13.7 3.7 0.29 0.39 2.61 1.77

49.2 32.3 12.5 4.3 0.21 0.17 1.93 1.53

49.7 32.7 12.3 3.8 0.21 0.18 1.84 1.53

105.5

93.4

51.9 28.6 14.3 3.8 0.3 0.2 1.6 2.0

37.9 22.4 11.2 3.1 0.2 0.1 0.5 1.4

P-Value Linear

Quadratic

4.5

0.45

0.86

48.7 32.4 12.2 3.4 0.41 0.29 3.87 1.56

1.4 1.9 1.1 0.4 0.10 0.11 1.03 0.14

0.26 0.25 0.37 0.45 0.43 0.59 0.44 0.32

0.71 0.38 0.62 0.25 0.20 0.16 0.21 0.34

87.9

103.6

17.9

0.89

0.46

52.3 28.4 14.3 3.5 0.4 0.4 1.0 2.0

50.1 31.2 13.7 4.3 0.3 0.2 0.4 1.6

6.2 5.4 2.2 0.7 0.1 0.1 0.4 0.4

0.76 0.58 0.91 0.58 0.84 0.57 0.74 0.11

0.37 0.42 0.60 0.29 0.62 0.79 0.70 0.54

CON: 0 mg β-extract or iso-α-extract/kg diet DM; Low: 600 mg/kg diet DM; MED: 1200 mg/kg diet DM; HIGH: 1800 mg /kg diet DM. Standard error of the mean, n = 4 replicates per treatment.

A:P ratio were observed after inclusion of BE. Moreover, total branched-chain VFA (BCVFA) were unaffected (P > 0.10) by BE inclusion. Dry matter and organic matter digestion were not affected (P > 0.10) by the inclusion of BE (Table 4). Additionally, BE did not affect (P > 0.10) the digestion of NDF or ADF. Ammonia nitrogen concentration was unaltered by BE inclusion, as were the flows of NH3-N, Non NH3-N, Microbial N and Dietary N (Table 5). Finally, CP degradation and EMPS were not affected by treatment (P > 0.10). Crude protein digestion and the efficiency of microbial protein synthesis (EMPS) were similar to previous reports in rumen continuous culture (Mansfield and Stern, 1994; Miller-Webster et al., 2002; Griswold et al., 2003). 3.2. Experiment 2: Iso-α-extract inclusion Iso-α-extract linearly increased (P = 0.04) mean pH but not (P > 0.10) minimum or maximum pH (Table 2). The time spent 5

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Table 4 Effects of increasing concentrations of hop β-extract or iso-α-extract on DM, OM and fiber digestion in continuous culture. Digestion

Treatmenta

SEMb

CON

LOW

MED

HIGH

Experiment 1 (β-extract inclusion) True DM digestion, % True OM digestion, % NDF digestion, % ADF digestion, %

58.1 55.4 43.8 42.6

55.7 54.1 39.3 35.6

55.0 52.9 39.8 37.9

56.3 54.0 40.0 41.0

Experiment 2 (Iso-α-extract inclusion) True DM digestion, % True OM digestion, % NDF digestion, % ADF digestion, %

57.1 51.4 36.4 42.4

58.6 52.4 35.7 39.5

58.4 53.0 43.0 48.0

57.0 51.0 34.4 39.5

a b

P-Value Linear

Quadratic

2.7 2.4 3.4 4.9

0.61 0.64 0.49 0.91

0.49 0.63 0.50 0.32

5.1 4.8 4.8 4.1

0.98 0.98 0.96 0.99

0.78 0.76 0.43 0.51

CON: 0 mg β-extract or iso-α-extract/kg diet DM; Low: 600 mg/kg diet DM; MED: 1200 mg/kg diet DM; HIGH: 1800 mg /kg diet DM. Standard error of the mean, n = 4 replicates per treatment.

Table 5 Effects of increasing concentrations of hop β-extract or iso-α-extract on nitrogen metabolism in continuous culture. Item

Experiment 1 (β-extract inclusion) NH3-N, mg/dL N Flow, g/d NH3-N Non NH3-N Microbial N Dietary N CP degradation, g/100 g EMPSd Experiment 2 (Iso-α-extract inclusion) NH3-N, mg/dL N Flow, g/d NH3-N Non NH3-N Microbial N Dietary N CP degradation, g/100 g EMPSc

Treatmenta

SEMb

CON

LOW

MED

HIGH

6.9

5.1

6.7

7.5

0.17 2.17 1.15 1.02 62.0 28.2

0.13 2.25 1.17 1.08 58.9 29.8

0.17 2.20 1.10 1.10 58.1 28.5

7.4

5.3

0.19 2.07 0.94 1.05 58.1 39.5

0.14 2.10 1.05 1.05 60.5 38.2

P-Value Linear

Quadratic

0.8

0.38

0.13

0.18 2.17 1.13 1.03 60.8 28.6

0.02 0.06 0.06 0.09 3.6 1.2

0.56 0.82 0.61 0.87 0.84 0.98

0.18 0.40 0.89 0.51 0.46 0.56

7.6

6.8

2.0

0.95

0.76

0.19 1.86 0.86 1.00 63.3 31.8

0.17 2.20 1.05 1.15 57.2 44.4

0.1 0.1 0.3 0.3 9.4 9.2

0.97 0.79 0.93 0.98 0.97 0.84

0.76 0.26 0.90 0.68 0.67 0.47

CON: 0 mg β-extract or iso-α-extract/kg diet DM; Low: 600 mg/kg diet DM; MED: 1200 mg/kg diet DM; HIGH: 1800 mg /kg diet DM. Standard error of the mean, n = 4 replicates per treatment. c EMPS: efficiency of microbial protein synthesis (g of microbial N/kg of OM truly digested). a

b

below pH 5.8 and time between pH 5.8 and 6.2 were not linearly affected (P > 0.10) by treatment. However, there was a tendency for a linear increase in time spent above pH 6.2 with increasing concentrations of IE. Iso-extract inclusion did not affect (P > 0.10) total VFA concentration or molar proportions of any individual VFA (Table 3). Dry matter and organic matter digestion were not modified (P > 0.10) by inclusion of IE, nor was the digestion of ADF or NDF (Table 4). Iso-α-extract did not affect (P > 0.10) the flows NH3-N, Non NH3-N, Microbial N and Dietary N, nor the NH3-N concentration within the fermenters (Table 5). Lastly, IE failed to impact (P > 0.10) CP degradation or EMPS.

4. Discussion Previous studies detected no changes in fermentation pH after the addition of whole hops or ensiled hops in the rumen (Al-Mamun et al., 2011; Narvaez et al., 2013a). Similarly, supplementation of hop extracts to batch culture incubations did not impact fermentation pH (Flythe and Aiken, 2010). Contrary to these results, we observed that increasing doses of either hops β-acid extract or iso-α-extract linearly increased fermentation pH within continuous culture fermenters. The changes in rumen pH due to BE or IE inclusion may be related to reducing the production of lactic acid. During beer production, one of the primary effects of hops is the reduction of lactic acid bacteria which cause beer spoilage (Suzuki et al., 2005). In our study, BE and IE may have also inhibited the growth of lactic acid producing bacteria, leading to increase in pH. 6

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While rumen pH was altered by treatment, VFA concentration and VFA profile were not altered by either BE or IE in experiments 1 and 2, respectively. These results contrast with previous reports suggesting changes in VFA profile due to addition of hops or hop acids. Flythe and Aiken (2010) detected a reduction in A:P ratio when hops β-extract was supplied at 30 or 60 ppm β-acids in batch culture. Narvaez et al. (2013a) also observed an increase in the concentration of propionate and a decrease in A:P ratio after addition of hops extract containing both α- (49.3 mg/g DM) and β- (37.5 mg/d DM) acids. In batch culture, two high α-acid hop varieties reduced total VFA as well as the molar proportions of acetate and butyrate (Lavrenčič et al., 2014). Effects of hops on A:P ratio was reported to be greater in high-concentrate diets compared with high-forage diets (Narvaez et al., 2011). The results of the current study agreed with observations by Al-Mamun et al. (2009), who detected no effect of hops on rumen production of total VFA or individual molar proportions of VFA. However, they did observe a tendency for increased plasma acetate. The contrast in results between batch culture and continuous culture or in vivo experiments may also be related to microbial adaptation to hop acids. While hop acids show strong effects in batch, pure and co-culture, these systems typically use short-term incubations, limiting time for potential microbial adaptation (Flythe, 2009; Narvaez et al., 2011). The ability of bacterial species to adapt to anti-microbial compounds is well-documented (Lohner and Blondelle, 2005; Delcour, 2009). In the brewing industry, several mechanisms of microbial adaptation to hops acids have been described, including induced expression of multi-drug transporters and a mechanism for energy conservation (Sami et al., 1997; Suzuki et al., 2002; Behr et al., 2007). Rumen microorganisms exhibit the ability to develop resistance to ionophores, specifically monensin, by increasing extracellular polysaccharides that prevent disruption of the cellular membrane (Callaway and Russell, 1999; Russell and Houlihan, 2003). In the current experiment, microbial adaptation was not examined, and the effects of hops were not measured until the post-adaptation period. Future experiments studying the effects of hops on rumen fermentation in continuous culture or in vivo models should examine both their short-term (0–2 days after treatment) and longer-term effects (> 2 days). In addition to no observed effects on VFA production, neither experiment 1 nor experiment 2 demonstrated any effects of hop acids on dry matter, organic matter, or fiber degradation. These results contrast with those observed by Narvaez et al. (2013a), who detected a reduction in true DM disappearance when extracts containing α- and β-acids at 22.5 and 30 ug/mL of inoculum (1.8 and 2.4 μg/kg substrate), respectively were added to rumen batch cultures. The differences between these experiments may be related to the length of exposure to hops and potential microbial adaptation. Additionally, relative concentrations of substrate, microbial inoculum, and hop bioactive compounds can vary widely from batch culture to continuous culture, further complicating the comparisons between studies. In vivo experiments in finishing steers demonstrated no effects of pelleted hops (Wang et al., 2010) or BE (Schmidt et al., 2006) on DM, NDF, or ADF digestibility. Furthermore, the discrepancy in these results may be related to differences in extract preparation and inclusion level between experiments. Crude protein digestion was also unaffected by increased concentrations of either BE or IE in experiments 1 and 2, respectively. Results contrast with observations by Flythe (2009), which showed reductions in hyper-ammonia producing bacteria by inclusion of 30 ppm BE in pure and co-culture. Whole hop varieties high in α-acids decreased CP degradation in batch culture incubated with rumen fluid from sheep (Lavrenčič et al., 2014). However, in vivo experiments have failed to show differences in ruminal NH3-N concentrations after BE addition (Drouillard et al., 2009). Disagreement between in vitro and in vivo data may be related to the influence of protozoa which are not present in pure and co-cultures, and are poorly maintained in continuous culture (Mansfield et al., 1995). Protozoa affect rumen nitrogen metabolism by sequestering microbial N via bacterial predation and slow rumen turnover (Koenig et al., 2000). This discrepancy may also be related to interactions between microbial species within natural rumens and continuous culture systems that are not present in pure or co-culture. Additionally, relative concentrations of substrate, microbial inoculum, and hop bioactive compounds can vary widely from batch culture to continuous culture, further complicating the comparisons between studies.

5. Conclusions Beta-acids and iso-alpha-acids alter fermentation within rumen continuous culture fermenters by increasing rumen pH. However, contrary to our hypothesis, these changes in pH were not connected with changes acetate to propionate ratio or ammonia-N concentration. The discrepancy between our experiment and batch culture experiments that observe changes in VFA and nitrogen metabolism due to hop acids may be due to microbial adaptation in long term culture. The results of this paper suggest that while hop extracts may have potential to mediate rumen pH, they do not appear to be a strong candidate to replace ionophore antibiotics as modifiers of rumen fermentation.

Funding Research was supported by Minnesota Agricultural Experiment Station (MAES) Project No. MIN-16-051: Impact of Factors that Affect Rumen Microbial Ecology.

Declaration of Competing Interest The authors declare no conflict of interest. 7

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Acknowledgements The authors gratefully acknowledge the technical assistance of I. Ceconi, H. Johnson, and W. Weber (University of Minnesota, St. Paul, MN). Gratitude is also expressed to B. Dayton and B. Crooker for use of laboratory equipment (University of Minnesota, St. Paul, MN). Authors also thank S.S. Steiner, Inc. for donation of hop extracts. Acknowledgement is given to the staff at the University of Minnesota Dairy Cattle Teaching and Research Center for care of cows used in the experiment. References Al-Mamun, M., Goto, K., Chiba, S., Sano, H., 2009. Responses of plasma acetate metabolism to hop (Humulus lupulus L.) in sheep. Int. J. Biol. Sci. 5, 287–292. https:// doi.org/10.7150/ijbs.5.287. Al-Mamun, M., Saito, A., Sano, H., 2011. Effect of ensiled hop (Humulus lupulus L.) residues on plasma acetate turnover rate in sheep. Anim. Sci. J. 82, 451–455. https://doi.org/10.1111/j.1740-0929.2010.00867.x. AOAC, 2005. Official Methods of Analysis, 18th ed. Association of Official Analytical Chemists, Arlington, VA. AOAC, 1990. Official Methods of Analysis, 15th ed. Association of Official Analytical Chemists, Arlington, VA. Behr, J., Israel, L., Gänzle, M.G., Vogel, R.F., 2007. Proteomic approach for characterization of hop-inducible proteins in Lactobacillus brevis. Appl. Environ. Microbiol. 73, 3300–3306. https://doi.org/10.1128/AEM.00124-07. Bergen, W.G., Bates, D.B., 1983. Ionophores: their effect on production efficiency and mode of action. J. Anim. Sci. 58, 1465–1483. https://doi.org/10.2134/jas1984. 5861465x. Callaway, T.R., Russell, J.B., 1999. Selection of a highly monensin-resistant Prevotella bryantii subpopulation with altered outer membrane characteristics. Appl. Environ. Microbiol. 65, 4753–4759. Delcour, A.H., 2009. Outer membrane permeability and antibiotic resistance. Biochim. Biophys. Acta (BBA)-Proteins Proteomics 1794, 808–816. https://doi.org/10. 1016/j.bbapap.2008.11.005. Drouillard, J.S., Garden, S.Q., Shelor, M.K., Higgins, J.J., Garden, S., 2009. Effects of beta acid extracts of hops on ruminal metabolism and apparent total tract digestibility by steers fed high concentrate diets. In: Chilliard, Y., Glasser, F., Faulconnier, Y., Bocquier, F., Veissier, I., Doreau, M. (Eds.), Ruminant Physiology: Digestion, Metabolism and Effects of Nutrition on Reproduction and Welfare:Proceedings of the XIth International Symposium on Ruminant Physiology. Wageningen Academic Publishers, pp. 164–165. FASS, 2010. Guide for the Care and Use of Agricultural Animals in Research and Teaching. Fessenden, S.W., Carpenter, A.J., Ruiz-Moreno, M., Jenkins, T.C., Stern, M.D., 2017. Effects of bismuth subsalicylate and dietary sulfur level on fermentation by ruminal microbes in continuous culture. Transl. Anim. Sci. 1, 559–569. https://doi.org/10.2527/tas2017.0062. Flythe, M., Harlow, B., Aiken, G., Gellin, G., Kagan, I., Pappas, J., 2017. Inhibition of growth and ammonia production of ruminal hyper ammonia-aroducing bacteria by Chinook or Galena hops after long-term storage. Fermentation 3, 68. https://doi.org/10.3390/fermentation3040068. Flythe, M.D., 2009. The antimicrobial effects of hops (Humulus lupulus L.) on ruminal hyper ammonia-producing bacteria. Lett. Appl. Microbiol. 48, 712–717. https:// doi.org/10.1111/j.1472-765X.2009.02600.x. Flythe, M.D., Aiken, G.E., 2010. Effects of hops (Humulus lupulus L.) extract on volatile fatty acid production by rumen bacteria. J. Appl. Microbiol. 109, 1169–1176. https://doi.org/10.1111/j.1365-2672.2010.04739.x. García-González, R., López, S., Fernández, M., Bodas, R., González, J.S., 2008. Screening the activity of plants and spices for decreasing ruminal methane production in vitro. Anim. Feed Sci. Technol. 147, 36–52. https://doi.org/10.1016/j.anifeedsci.2007.09.008. Griswold, K.E., Apgar, G.A., Bouton, J., Firkins, J.L., 2003. Effects of urea infusion and ruminal degradable protein concentration on microbial growth, digestibility, and fermentation in continuous culture. J. Anim. Sci. 81, 329–336. https://doi.org/10.2527/2003.811329x. Haas, G.J., Barsoumian, R., 1994. Antimicrobial activity of hop resins. J. Food Prot. 57, 59–61. https://doi.org/10.4315/0362-028X-57.1.59. Hannah, S.M., Stern, M.D., Ehle, F.R., 1986. Evaluation of a dual flow continuous culture system for estimating bacterial fermentation in vivo of mixed diets containing various soya bean products. Anim. Feed Sci. Technol. 16, 51–62. https://doi.org/10.1016/0377-8401(86)90049-0. Koenig, K.M., Newbold, C.J., McIntosh, F.M., Rode, L.M., 2000. Effects of protozoa on bacterial nitrogen recycling in the rumen. J. Anim. Sci. 78, 2431. https://doi. org/10.2527/2000.7892431x. Krishna, G., Czerkawski, J.W., Breckenridge, G., 1986. Fermentation of various preparations of spent hops (Humulus lupulus L.) using the rumen simulation technique (RUSITEC). Agric. Wastes 17, 99–117. https://doi.org/10.1016/0141-4607(86)90049-1. Lavrenčič, A., Levart, A., Košir, I.J., Cerenak, A., 2014. Influence of two hop (Humulus lupulus L.) varieties on in vitro dry matter and crude protein degradability and digestibility in ruminants. J. Sci. Food Agric. 94, 1248–1252. https://doi.org/10.1002/jsfa.6407. Lenth, R.V., 2007. Statistical power calculations. J. Anim. Sci. 85, E24–E29. https://doi.org/10.2527/jas.2006-449. Lohner, K., Blondelle, S.E., 2005. Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics. Comb. Chem. High Throughput Screen. 8, 241–256. Mansfield, H.R., Endres, M.I., Stern, M.D., 1995. Comparison of microbial fermentation in the rumen of dairy cows and dual flow continuous culture. Anim. Feed Sci. Technol. 55, 47–66. https://doi.org/10.1016/0377-8401(95)98202-8. Mansfield, H.R., Stern, M.D., 1994. Effects of soybean hulls and lignosulfonate-treated soybean meal on ruminal fermentation in lactating dairy cows. J. Dairy Sci. 77, 1070–1083. https://doi.org/10.3168/jds.S0022-0302(94)77042-9. Miller-Webster, T., Hoover, W.H., Holt, M., Nocek, J.E., Mitchell, G.E., 2002. Influence of yeast culture on ruminal microbial metabolism in continuous culture. J. Dairy Sci. 85, 2009–2014. https://doi.org/10.3168/jds.S0022-0302(02)74277-X. Narvaez, N., Wang, Y., McAllister, T., 2013a. Effects of extracts of Humulus lupulus (hops) and Yucca schidigera applied alone or in combination with monensin on rumen fermentation and microbial populations in vitro. J. Sci. Food Agric. 93, 2517–2522. https://doi.org/10.1002/jsfa.6068. Narvaez, N., Wang, Y., Xu, Z., McAllister, T., 2011. Effects of hops on in vitro ruminal fermentation of diets varying in forage content. Livest. Sci. 138, 193–201. https://doi.org/10.1016/j.livsci.2010.12.028. NRC, 2001. Nutritional Requirements of Dairy Cattle, 7th rev ed. National Academy Press, Washington, DC. Russell, J.B., Houlihan, A.J., 2003. Ionophore resistance of ruminal bacteria and its potential impact on human health. FEMS Microbiol. Rev. 27, 65–74. https://doi. org/10.1016/S0168-6445(03)00019-6. Sakamoto, K., Konings, W.N., 2003. Beer spoilage bacteria and hop resistance. Int. J. Food Microbiol. 89, 105–124. https://doi.org/10.1016/S0168-1605(03)00153-3. Sami, M., Yamashita, H., Hirono, T., Kadokura, H., Kitamoto, K., Yoda, K., Yamasaki, M., 1997. Hop-resistant Lactobacillus brevis contains a novel plasmid harboring a multidrug resistance-like gene. J. Ferment. Bioeng. 84, 1–6. https://doi.org/10.1016/S0922-338X(97)82778-X. Schmidt, M.A., Nelson, M.L., Michal, J.J., Westberg, H.H., 2006. Effects of hop acids. II. Beta acids on ruminal methane emission, protozoal population, fermentation, and CoM concentration in cannulated finishing steers. J. Anim. Sci. 84, 240. Srinivasan, V., Goldberg, D., Haas, G., 2004. Contributions to the antimicrobial spectrum of hop constituents. Econ. Bot. 58, S230–S238. https://doi.org/10.1663/ 0013-0001(2004)58[S230:CTTASO]2.0.CO;2. Storlien, T.M., Harstad, O.M., Narvaez, N., Wang, Y., McAllister, T.A., 2012. Effects of different oils and plant extracts on in vitro ruminal methane production. Acta Agric. Scand. Sect. A – Anim. Sci. 62, 300–304. https://doi.org/10.1080/09064702.2013.773058. Suzuki, K., Iijima, K., Ozaki, K., Yamashita, H., 2005. Isolation of a hop-sensitive variant of Lactobacillus lindneri and identification of genetic markers for beer spoilage ability of lactic acid bacteria. Appl. Environ. Microbiol. 71, 5089–5097. https://doi.org/10.1128/AEM.71.9.5089-5097.2005.

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I.J. Salfer, et al.

Suzuki, K., Sami, M., Kadokura, H., Nakajima, H., Kitamoto, K., 2002. Biochemical characterization of horA-independent hop resistance mechanism in Lactobacillus brevis. Int. J. Food Microbiol. 76, 223–230. https://doi.org/10.1016/S0168-1605(02)00016-8. Van Soest, P.Jvan, Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. https://doi.org/10.3168/jds.s0022-0302(91)78551-2. Wang, Y., Chaves, A.V., Rigby, F.L., He, M.L., McAllister, T.A., 2010. Effects of hops on ruminal fermentation, growth, carcass traits and shedding of Escherichia coli of feedlot cattle. Livest. Sci. 129, 135–140. https://doi.org/10.1016/j.livsci.2010.01.015. Weller, R.A., Pilgrim, A.F., 1974. Passage of protozoa and volatile fatty acids from the rumen of the sheep and from a continuous in vitro fermentation system. Br. J. Nutr. 32, 341–351. https://doi.org/10.1079/bjn19740087. Zinn, R.A., Owens, F.N., 1986. A rapid procedure for purine measurement and its use for estimating net ruminal protein synthesis. Can. J. Anim. Sci. 66, 157–166. https://doi.org/10.4141/cjas86-017.

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