The Antibacterial Effect of Humulus lupulus (Hops) against Mycobacterium bovis BCG: A Promising Alternative in the Fight against Bovine Tuberculosis?

: The female ﬂowers of the Humulus lupulus plant or Hops have been used extensively within the brewing industry for their aroma and bitterness properties. It was also found that beer that contained hops was less likely to spoil, thus revealing the antimicrobial potential of these plants. One species of bacteria, Mycobacterium spp., is of particular interest as it is the causative agent of both human and animal forms of tuberculosis (TB). In this study an aqueous extraction process was employed to analyse the antibacterial properties of 50 hop extracts (45 individual variants); against M. bovis BCG. Using an agar well diffusion assay we found that all hops exhibited a level of inhibitory activity which ranged from 1.2 mm (+/ − 0.08 mm) in the case of hop variant; Target, to 15.7 mm (+/ − 0.45 mm) in the case of hop variant Citra. The Citra variant had a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 16% v / v . This is the ﬁrst study to analyse a wide range of hops for their antimicrobial potential against M. bovis BCG and recommends that further research focuses on other Mycobacteria spp., the potential for antimicrobial synergy and the antibacterial effect of individual components.


Introduction
Plants and their derivatives represent a diverse resource from which discovering new, efficient and safe antibiotics is a priority.Research is currently focusing on novel sources and early pharmacopeias' to discover plants or extracts which were once considered effective but perhaps fell out of favour with the introduction of antibiotics.The plant Humulus lupulus, known as the hop, is a potential source of novel antimicrobial activity [1].
The hop plant itself has been employed in the brewing industry due to its positive effect on flavouring and aroma, its perhaps less known for its antibacterial properties against a range of clinical and food related microorganisms such as Listeria monocytogenes [2].The antibacterial effect can be attributed in part to the presence of humulone (alpha acid); lupulone (beta acid) and the polyphenol xanthohumol [2].The addition of hops in the beer brewing process has been observed to prevent beer spoilage when compared to its 'unhopped' counterpart [3].Current research has focused on developing the commercial potential of hops such as: The use of specific hop extracts to improve the aroma and flavour of beer [4], The acceleration of lautering [5], reduction of methane emissions [6] and as a food preservative [2].
Given the emergence of multidrug resistant strains of Mycobacterium [7][8][9][10].Several studies have investigated the role of the hop plant as a treatment or preventative agent against Mycobacterium tuberculosis infection [11][12][13].Serkani  highlighting the potential for hops or their extracts to be exploited for their antimicrobial potential, particularly against mycobacterium spp.[12].
A related organism to M. tuberculosis is M. bovis which is responsible for the animal form of the disease; Bovine tuberculosis (bTB).Current legislation prevents the use of antimicrobials (without written consent of the Welsh Government (Wales) [14] and The secretary of State (England) [15] in cattle either as a treatment or preventative measure, the only option is for infected animals to be culled (TB in Wales 2010) [16].Further to this, current European legislation has been updated to reduce the use of antibiotics in animals as growth promotors (Regulation (EU) 2019/6 of the European Parliament and of the Council of 11 December 2018 on veterinary medicinal products and repealing Directive 2001/82/EC).The legislation which came into force in January 2022 prevents the routine administering of antibiotics in groups of animals, bans the preventive use of antimicrobials via medicated feed [17].
In Wales, UK, between March 2020 and March 2021, 10,258 cattle were slaughtered for TB control which was a 11% decrease on the previous 12 months when 11,559 cattle were slaughtered, bovine infection with this pathogen is estimated to cost the UK economy over £100 million per year [16].
M. bovis, similar to M. tuberculosis, is also classed as a Category III pathogen and replicates at a similar rate making it an impractical bacterium to use in high through put screening assays.In contrast, the vaccine strain of M. bovis, Mycobacterium bovis Bacillus Calmette-Guérin [BCG) rarely causes disease in healthy individuals and replicates in the laboratory at a similar rate to M. smegmatis [18].In vitro studies have shown that BCG has a similar level of antibacterial sensitivity to anti-tuberculosis compounds making it a suitable model organism with which to screen for antibacterial activity [19,20].
In this study the inhibitory effect of fifty aqueous hop extracts against Mycobacterium bovis BCG was investigated; Hops were purchased as either pelleted varieties or as whole (unmacerated) hops, the alpha acid content was determined by the supplier.A previously optimized aqueous extraction procedure was undertaken to increase bioactive component extraction and agar-based methods were used to determine overall inhibitory effects and minimum inhibitory [MIC) and bactericidal concentrations (MBC) were identified.

Materials and Methods
All materials were purchased from Fisher scientific, UK unless otherwise stated.Microorganisms: Mycobacterium bovis BCG (Bacillus Calmette-Guerin) NCTC 5692 was employed in this study.The strain was purchased from the Health Protection Agency, Culture Collections, UK.The culture was stored in 10% glycerol on Microbank™ Cryoprotective beads (Pro-Lab Diagnostics Ltd., Birkenhead, UK) at −80 • C until required.
General laboratory media: All laboratory media and reagents were prepared as per manufacturer's instructions and were sterilised at 121 • C for 15 min at 15 PSI.All analyses were performed in triplicate on at least three occasions.Middlebrook 7H9 Broth and 7H10 Agar (Sigma, UK) were used for the propagation of M. bovis BCG NCTC 5692.
Hops: Hops were purchased from the Malt Miller Ltd All hops were delivered at room temperature in vacuum packaging and were protected from light exposure.Once opened, hop material was stored at 3 • C in the dark until extraction, typically this was within 24 h.In Table 1 the hop variants employed in the study along with their level of processing (whole hop cones or pelleted cones) and the stated alpha acid content (% w/w) is detailed.
Aqueous Hop Extraction Method: Upon delivery, hop material was stored at 3 • C within an airtight container.Prior to extraction, 5 g of hop material was mechanically macerated and passed through a filter with pore size of <1 mm and suspended, with mixing at 30 RPM, in 200 mL of sterile deionized water and heated at 100 • C for 60 min.This method was previously optimized by the authors and is detailed in [13].The hop solutions were allowed to cool prior to coarse filtration using a 1 mm gauze.The resultant solution was termed the hop extract.
Bacterial standardisation: A fresh streak plate agar culture of M. bovis BCG NCTC 5692 was prepared and incubated at 37 • C for 72 h and a subculture of a single colony was transferred to the appropriate broth for the same duration.The culture was centrifuged at 3000 RPM for 15 min at 4 • C. The supernatant was removed via aspiration and the resultant bacterial pellet resuspended in sterile phosphate buffered saline (PBS).An optical density-aerobic colony count was performed at 600 nm (Jenway 7300 spectrophotometer).In all tests the concentration of test microorganisms was adjusted to approximately 1 × 10 6 CFU/mL −1 using PBS.
Agar Well Diffusion Assay: Middlebrook 7H10 were prepared as previously described and allowed to cool to 40 • C. The agar was then gently inverted, and 20 mL aliquots were decanted into 45 × 90 mm petri dishes which were then allowed to cool and set within a laminar flow cabinet.
Plates were then inoculated with 150 µL of a bacterial suspension which had been standardised to approximately 1 × 10 6 CFU/mL −1 using a spectrophotometer, an even spread was made across the plate using a sterile flat sided spreader, plates were then incubated statically at 37 • C for 3 h.A previously sterilised 6 mm borer was then used to create 4 equally distanced wells per plate [13].
For each hop extract or test bacterium, newly sterilised equipment was used.In each well 80 µL of the aqueous hop extract was pipetted; in addition to this, on each plate 80 µL of H 2 O was also pipetted into a well as a negative control and 80 µL of oleic acid (Merck Life Science UK Ltd, Gillingham, UK) at a concentration of 8 µg mL −1 in 50% DMSO as a positive control.Plates were transferred to a static 37 • C incubator (Thermo Fisher Scientific, Newport, UK) for 72 h.Inhibition of bacterial growth was observed by a clear zone of inhibition around the test well.The size of the zone of inhibition was determined by measuring two diameters at right angles across the zone and subtracting the diameter of the well itself (6 mm).Each assay was performed on at least 3 separate occasions in triplicate on each bacterial species with each hop extract [13].
Determination of the minimum inhibitory and minimum bactericidal concentration (MIC and MBC): Agar was produced at double its standard concentration (Middlebrook 7H10 19 g 450 mL −1 diH 2 O), autoclaved and allowed to cool to 40 • C. Aqueous hop extracts, sterile distilled water and molten agar where then added to a 24 well plate (Thermo Fisher Scientific, Newport, UK) and were mixed via pipette aspiration to give a final hop extract concentration between 0 and 33% (v/v) as detailed in Table 2.
After cooling, the plate was transferred to a static incubator for 3 h at 37 • C. A bacterial suspension was then standardised to 1 × 10 6 cfu mL −1 and two 20 µL aliquots were pipetted onto the surface of each well.Plates were then incubated for 72 h.
The MIC was determined by visually inspecting each well after incubation, the lowest concentration of hop extract which inhibited growth, was considered the MIC.To determine the minimum bactericidal concentration (MBC) the surface of each well was swabbed with a sterile swab and transferred to 10 mL Middlebrook 7H9 broth [13].
The inoculated media was then incubated with shaking at 150 rpm at 37 • C for 72 h.After incubation, the absorbance of each solution was read at 600 nm (UV -6010, Thermo Fisher Scientific, Newport, UK), using sterile broth as a negative control, the lowest concentration of hops that prevented the growth of bacteria was considered the minimum bactericidal concentration (MBC) determined by a 0-absorbance reading compared to the control at 600 nm.
The method was repeated on at least 3 separate occasions in triplicate for each test bacteria unless otherwise specified.

Results
We analysed all hop extracts (n = 50) against M. bovis BCG.The least active hop extract was determined to be Target whole hop (Average zone of inhibition 1.2 mm (+/− 0.08 mm)) whilst the greatest effect was observed to be Citra pellets (15.7 mm (+/− 0.45 mm)).It was interesting to note that all hops exhibited some form of inhibitory effect against the test bacterium (Figure 1).Previous studies [21,22] have suggested that the alpha acid content is linked to antibacterial activity, in Figure 2 the alpha acid content (as determined by the supplier) was plotted against the average zone of inhibition.Previous studies [21,22] have suggested that the alpha acid content is linked to antibacterial activity, in Figure 2 the alpha acid content (as determined by the supplier) was plotted against the average zone of inhibition.Previous studies [21,22] have suggested that the alpha acid content is linked to antibacterial activity, in Figure 2 the alpha acid content (as determined by the supplier) was plotted against the average zone of inhibition.Although no direct correlation was observed between the stated alpha acid content and the average zone of inhibition; The MIC and MBC were next investigated as these are generally more sensitive methods for the determination of antimicrobial activity (Table 3).Although no direct correlation was observed between the stated alpha acid content and the average zone of inhibition; The MIC and MBC were next investigated as these are generally more sensitive methods for the determination of antimicrobial activity (Table 3).

Discussion
In this study it was observed that all aqueous hop extracts had an inhibitory effect against the growth of Mycobacterium bovis BCG; The MIC and MBC for each hop was determined and it was found that the Citra hop variant had the greatest level of antimicrobial activity.
The use of hops as an antimicrobial has been well documented, in Table 4 some of the current and pertinent published literature regarding the study of hops, their extracts (including commercial CO 2 extracts) and their individual components such as humulone, lupulone and xanthohumol in vitro against bacteria, viruses, fungi and their potential for biofilm disruption and in rumen applications can be observed.Table 4.An overview of recent and pertinent antimicrobial, antifungal, antiviral and antibiofilm studies concerning the use of hops and their extracts.The authors found no references to studies that investigated the antimicrobial effect of hops effect against Mycoacterium bovis.It was interesting to note from Table 4 that although there are a number of studies which investigated the antimicrobial effect of hops against mycobacteria [11][12][13]25] and as additives in ruminant feedstuffs [31] or for their potential use in methane reduction [6], there were no studies which investigated the use of hops against Mycoacterium bovis.

Type of
To the best of our knowledge this is the first study to investigate the inhibitory effect of such a wide range of hop extracts against Mycobacterium bovis.M. bovis can cause tuberculosis in cattle (bovine tuberculosis (bTB)).bTB is an endemic, zoonotic disease commonly seen within cattle.In the UK, bTB is especially prevalent in Wales and the South West of England.Between December 2020 and December 2021 there were 665 new herd incidents (positive bTB cases) in Wales an increase of 8.5% compared to December 2019-2020 [16].As previously mentioned, the requirement to cull infected animals produces a large economic burden for farmers in these circumstances, an alternative control method includes the use of culling potential animals such as badgers which are purported to be the main transmission route between cattle is often met with public controversy [32].Therefore, a potential preventative approach using natural products such as hops may be a promising alternative to animal slaughter.
We firstly investigated the antimicrobial potential of each hop variant using an agar well diffusion assay.Although not specific, this method is a useful screening tool for large scale antimicrobial detection [33].It was interesting to note that in all hop extracts we observed some level of inhibitory activity which ranged between 'Target' at 1.2 mm (+/− 0.08 mm) to Citra pellets at 15.7 mm (+)/− 0.45 mm).It was also observed that in all cases the pelleted version of the variant had a greater observed antimicrobial effect than its whole hop counterpart.This could be due to the reduced particle size of the hop material produced as a result of the pelleting process leading to increased extraction of bioactive components due to increased surface area; It could also be attributed to the compressed nature of the pellet reducing the potential for component oxidation or degradation due to oxygen exposure and indeed a larger amount of hop material being required to manufacture a single hop pellet [34,35].
Whilst we acknowledge that the agar well diffusion assay may not be as sensitive as the broth microdilution method, it has been shown to be a useful tool in the determination of overall antimicrobial activity [33].The limitations of the method can be attributed to differences in bacterial concentration, agar volume and sample addition, to ensure comparability these were kept constant in our analysis.Due to the turbidity of the hop extracts utilized in the study we did not employ a spectrophotometric method for the determination of the minimum inhibitory and bactericidal concentrations.Instead, we relied upon an agar-based method where the agar and hop extract were homogenized to form a known concentration.Further studies would be improved by increased filtration of the hop extracts to remove large organic matter prior to analysis as demonstrated by Gregory and colleagues [21].
We found that in the majority of our extracts the minimum inhibitory and bactericidal concentrations exceeded our limit of detection (33% v/v).However, in correlation with the zone of inhibition results from 13 variants we observed an MIC range between 16% v/v (Citra Pellets) and 26% v/v (Apollo whole hops).The MBC ranged between 16% v/v (Citra Pellets) and 26% v/v (Northern Brewer).The pelleted version of the Citra variant exhibited a higher zone of inhibition and lower MIC/MBC compared to the whole hop variant.This could be attributed to the pelleting process which macerates hop material and increases surface area for extraction [34,35].
The antimicrobial activity of hops has previously been linked to the hop alpha acid humulone, beta acid lupulone and polyphenol xanthohumol [22,25].Gerhauser (2005) demonstrated the antibacterial activity of xanthohumol against the Gram-positive microorganisms Staphylococcus aureus and Streptococcus mutans along with antiviral and antiparasitic activity [36].
In our study, there was no observed link between the stated alpha acid content and the antimicrobial activity against Mycobacterium bovis BCG.It may be the case however that the extraction method which is known to lead to isomerisation of such compounds may have a detrimental effect on these components [37] although isohumulone and xanthohumol can be considered important hop bioactive compounds [22,25,28].
Future research could focus on the characterization of the antibacterial components of the hop extracts utilized in this study.Prencipe and colleagues (2014) developed a high-performance liquid chromatography method for the bioactive compounds within hops in combination with a range of extraction techniques to improve secondary metabolite identification which may improve identification of future antimicrobial components in similar studies [38].It may be the case that the antimicrobial effect is not due to the presence of a single component found within the hop extract but is perhaps a synergistic combination of different components.We also acknowledge that at this stage we have not assessed the effect of these particular extracts at the tested concentrations for their cell toxicity.Ensuring the safety of such extracts for potential therapeutic use would be pertinent in future studies and would further point towards the basis of antimicrobial activity [39].
In this study we observed that the Citra hop variant had the greatest amount of antimicrobial activity against M. bovis.The Citra hop variant also known as 'HBC 394 ′ was patented by inventors Eugene Probasco and Jason Perrault as a product of a control breeding program by Hop Breeding Co LLC.The variety is a cross between female parent '8801-02 ′ ('Hallertauer mittlefrueh' × '853-144M') (not patented) and male parent '8801-01M ('Hallertauer mittlefrueh' × '853-144M') (not patented).In general, the Citra variety produces approximately 11-13% Alpha Acids, 3.4-4.5%Beta acids and 22-24% Cohumulone [40].The alpha acid concentration is slightly lower than that reported by our supplier.It has been previously reported that weather conditions during growing can affect the alpha acid content [41] along with harvesting following flowering [42]; In tea it has been that catechin contents is affected by environmental effects such as growing altitude [43].Therefore, future similar studies could standardize the growth conditions and undertake alpha acid, beta acid and xanthohumol concentration analysis prior to testing.
Given the potential bioactive properties of hops against Mycobacteria spp.Future research could investigate the ability of hop extracts to inhibit the growth of other mycobacterial species such as Mycobacterium tuberculosis or Mycobacterium abscessus.Additionally, previous studies (Table 4) have explored the use of hops and their extracts as a potential feed addition for cattle to decrease methane production and found that hops and their extracts have an in vitro inhibitory effect on Methanobrevibacter ruminantium the main methane emitting microorganisms within ruminants [6].Similar studies such as those undertaken by Narvaez and colleagues (2011) have investigated the use of hop additions in ruminal fermentation and concluded that the addition of hop extracts may offer a means of decreasing ruminal methane emission without compromising the fermentability of feed [31].The caveat being that these studies have been completed in vitro and thus more work needs to be undertaken to understand potential in vivo effects.Given the ability of hops to inhibit Mycobacterium bovis and the potential to also reduce methane production the addition of certain hop components within cattle feed is an area that we believe justifies more research.
Hops and other plants such as tea [44], seaweed [45] and dandelions [46] offer potential solutions to the growing threat of antimicrobial resistance [47].
These plants offer the potential to be explored for antimicrobial synergistic activity; Natarajan and colleagues (2008) investigated the use of hop derived compounds such as lupulone and xanthohumol in combination with a range of antibiotics against both Grampositive and Gram-negative microorganisms using agar-based methods [48].A review by Fahle and colleagues (2022) highlighted the potential for hop components to be used with thirteen antibiotics and found effectiveness against both Gram-positive and Gram-negative microorganisms [49].
Within our own research we found that the majority of studies related to in vitro effects of hop components.Whilst this is promising, future studies could investigate the cellular effect of hop components and the antimicrobial effect in vivo.

Conclusions
This study characterized the antimicrobial effect of 50 different hop variants against Mycoabcteroum bovis BCG.All hop variants demonstrated inhibition against the microorganism, with the Citra hop variant having the lowest MIC and MBC.Previous studies have also investigated the potential for hops to reduce methane production in ruminants; the ability to both have a protective effect against bovine tuberculosis whilst reducing methane production is an area we recommend for future research.Clinically, future studies could include other Mycobacteria spp.along with the potential to include synergistic activity with antimicrobials as well as determining the toxicity of potential bioactive components.

Figure 1 .
Figure 1.The average zone of inhibition of aqueous hop extracts (n = 50) against M. bovis BCG.Experiments are the results of 3 replicates on three occasions +/− standard deviation.(p = Pelleted variant; W = Whole; unstated = whole).

Figure 1 .
Figure 1.The average zone of inhibition of aqueous hop extracts (n = 50) against M. bovis BCG.Experiments are the results of 3 replicates on three occasions +/− standard deviation.(p = Pelleted variant; W = Whole; unstated = whole).

Figure 2 .
Figure 2. Correlation of the average zone of inhibition against M. bovis BCG and the stated alpha acid content of the hop variants.

Figure 2 .
Figure 2. Correlation of the average zone of inhibition against M. bovis BCG and the stated alpha acid content of the hop variants.
and colleagues observed that drug resistant strains of M. tuberculosis were inhibited by exposure to hop extracts

Table 1 .
The hop variants employed in this study with stated alpha acid content (as determined by supplier).Hop material was supplied either as whole hops or pelleted hops under vacuum and stored at 3 • C until extraction.

Table 2 .
The volumes of double concentrated agar, SDW and aqueous hop extract used to generate a range of hop concentrations between 0 and 33% (v/v) within the agar incorporation assay.

Table 3 .
The MIC and MBC (%v/v) of 14 hop variants against M. bovis BCG as determined by the agar incorporation assay.All other hop variants exhibited a similar profile to 'Pacific Gem' with an MIC and MBC exceeding 33%.