Antimicrobial Activity of Chemical Hop (Humulus lupulus) Compounds: A Systematic Review and Meta-Analysis

1. Introduction

Humulus lupulus, commonly known as hop, is a dioecious climbing plant that belongs to the Cannabinaceae family, primarily cultivated for its critical role in the brewing industry [1]. Female flowers develop into cone-like structures called hops, which contain lupulin glands. These glands are rich in secondary metabolites such as polyphenols, bitter acids, and essential oils that contribute significantly to the flavor and aroma of beer, while they act as preservatives as well [1].

Beyond brewing, hops are increasingly investigated for their pharmacological properties. The key biochemical components of hops include primary metabolites, which support basic plant functions, and secondary metabolites that can influence ecological interactions of the plant [2]. The most notable secondary metabolites include polyphenols (e.g., xanthohumol, isoxanthohumol, catechin), bitter acids (humulone, cohumulone, lupulone, colupulone), and essential oils (myrcene, caryophyllene) [3,4,5,6]. Polyphenols, and partciularly prenylated flavonoids, exhibit strong antioxidant and antimicrobial activities [3,5]. Bitter acids undergo isomerization during wort boiling to form iso-α-acids, which are primarily responsible for beer's bitterness [4]. Essential oils provide the characteristic aroma of beer and are extracted through hydrodistillation [7].

The antimicrobial activity of hops extends to bacteria, fungi, and parasites, primarily through prenylated flavonoids and bitter acids [1,8]. α- and β-acids have been shown to inhibit Gram-positive bacteria such as Staphylococcus and Streptococcus species [9] while xanthohumol and 6-prenylnaringenin exhibit antifungal and antiparasitic effects [9]. Furthermore, xanthohumol has demonstrated anticancer potential by targeting signaling pathways such as Akt and NF-κB, which are involved in cancer cell proliferation, survival, and metastasis [8,10]. Additional health benefits include anti-obesity effects through enhanced lipolysis and beta-oxidation, dermatological benefits such as skin protection and anti-aging, and hepatoprotective effects against liver diseases [11].

Antimicrobial assays are fundamental in evaluating the therapeutic potential of botanical extracts, particularly in the search for novel compounds to combat antimicrobial resistance. These assays, which include disk diffusion, broth microdilution, and agar dilution methods, assess the inhibitory activity of crude plant extracts, fractions, and purified phytochemicals against a wide range of microorganisms [1,12,13,14]. Studies on Humulus lupulus have employed these methods to test various extract types - including aqueous, ethanolic, and supercritical CO₂ extracts (SFE) - alongside isolated compounds like xanthohumol and humulone [14]. This diversity of experimental directions were undertaken with a view to allowing researchers to determine the spectrum and mechanism of antimicrobial action, as well as the influence of extraction methods on bioactivity. Although standardized, in vitro assays provide reproducible and comparable data necessary for identifying promising bioactive candidates for pharmaceutical or food preservation applications do exist, experimental protocols in terms of concentrations and serial dilutions, incubation time and temperature, utilization of a wide spectrum of bacteria species and strains, are still to be standardized.

The objective of the present study is to systematically evaluate the antimicrobial efficacy of Humulus lupulus by synthesizing the available evidence through a robust, statistically rigorous approach. By conducting a meta-analysis of peer-reviewed studies, we aim to consolidate findings across diverse experimental contexts and identify key trends in the antimicrobial potency of hops. Specifically, we investigate the spectrum of activity against all bacterial strains tested to date, with a special emphasis on food spoilage microorganisms - organisms of significant relevance to public health and food industry sustainability. Furthermore, we examine the possibility of a potential probiotic sparing capacity of hop-derived compounds, a crucial consideration in preserving gut microbiota balance during antimicrobial treatment. Our study is framed within the principles of evidence-based practice, which emphasize the integration of comprehensive scientific evidence with clinical and industrial relevance to inform decision-making [15,16,17]. This synthesis offers a valuable foundation for future applied research on formulating strategies involving the value of hop phytoconstituents and extracts in food, cosmetic, and pharmaceutical sectors.

2. Materials and Methods

2.1. Literature Search Strategy and Eligibility Criteria of Selected Studies

The literature search was conducted in PubMed database to retrieve all potential research articles relevant to hop antimicrobial activity in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [18]. The literature search was conducted on 2/3/2024 using the keywords (hop or hops or "humulus lupulus") and (antimicrobial or antifungal or antibacterial or chemoprevention or biofilm or antiparasitic or antiviral) and all chemical compounds found in hop extracts. To include all possible relevant articles the reference lists of the selected articles were also scrutinized. Three researchers (DK, EA, and PK) independently assessed the search results. Any discrepancies in the initial evaluations were resolved through discussion with two additional reviewers (PB and GB).

Eligible studies had to contain MIC values of hop compounds or extracts tested on microorganisms. No language restrictions were imposed to reduce the risk of publication bias related to grey literature [19]. Titles and abstracts of retrieved articles were screened, and relevant articles were assessed for inclusion/exclusion criteria.

2.2. Data Extraction

Data extraction from eligible studies was performed on a Microsoft Excel sheet. Search results were extracted independently by two researchers (DK and EA). Selected studies contained data on concentrations of Humulus lupulus extracts and compounds against different microorganisms. Data extracted included PubMed ID, first author’s last name, publication year, MIC values, along with their SD or SE values, bacteria species and strains, names compounds, types of extracts, along with experimental conditions such as incubation time and temperature, Gram- classification (positive or negative), oxygen requirement (aerobic or anaerobic), and number of experimental replications. For studies reporting only the standard deviation (SD), the number of replicates was used to calculate the standard error of the mean (SEM) as follows: $SEM={\displaystyle \frac{SD}{\sqrt{n}}}.$ If neither SD nor SEM was provided, the missing values were imputed using the highest SD reported among studies using the same compound and microorganism [20].

2.3. Statistical Analysis

In the present meta-analysis, MIC values were used as effect size. Data were pooled using a random-effects meta-analysis [21] with inverse-variance weighting. MIC values and their 95% confidence intervals (CIs) were calculated for each compound across bacteria species, and incubation times.

Stratification according to classification of hop compounds and extracts into sub-categories was performed to infer possible parameters of their bioactivity. Stratifications according to their effect on food preseevation characteritics or health impact on humans (food spoilage / non-food spoilage or probiotic / non-probiotic) were also performed. To evaluate the impact of each individual study on the overall meta-analysis outcome, an influential analysis was conducted by sequentially excluding one study at a time and recalculating the statistical significance. Meta-analysis was performed with the statistical software Stata version 13.1, setting p-value for statistical significance less than or equal to 0.05 [22].

3. Results

3.1. Studies’ Selection and Characteristics

The literature search, following PRISMA guidelines, for antimicrobial activity of Humulus lupulus led to the retrieval of 2553 articles. After application of eligibility criteria, we resulted in 18 articles that included 450 individual studies (Figure 1).

The included studies were investigating the antimicrobial activity of various hop compounds (Figure 2) that include flavonoids, bitter acids, and three types of extracts, i.e. supercritical fluid extracts (CO₂-based, SFE), hydroalcoholic, and hydroacetonic. From them, 122 report MIC values on flavonoids, 152 on bitter acids, 67 studies on CO₂-extracts, 79 on hydralcoholic extracts and 28 on hydroacetonic extracts. Strains, which are used to measure antimicrobial activity against them, are 55. From the 450 studies, 174 of them were investigating antimicrobial effects after treatment for 24 hours, while 276 studies reported results for the 48 hours time point (Table 1). The characteristics of the included studies reporting MIC values, incubation time, and experimental repetitions were taken into consideration for the meta-analysis.

3.2. Meta-Analysis of MIC Values of Classes of Hop Compounds and Extracts

A persistent challenge in evaluating the antimicrobial activity of hop-derived extracts lies in the considerable variability introduced by multiple factors. One major concern is determining which bacterial species and strains most accurately reflect the true antimicrobial potential, as susceptibility can vary widely even within a single species. At the same time, hop extracts contain a complex mixture of bioactive compounds, each present at different concentrations that can be significantly influenced by the genetic background [40], the extraction method and the solvent used. These variables complicate interpretation, as changes in extraction parameters may favor the presence of certain compounds while minimizing others.

Therefore, we chose to conduct a meta-analysis and include a broad panel of all bacterial strains ever tested for hop compounds or extracts. To better understand their specific contributions to antimicrobial activity, we stratified our meta-analysis according to the two major different classes of natural compounds, the flavonoids, that are polyphenols, and the bitter acids that are prenylated acylphloroglucinol derivatives. As shown in Figure 3A, Table 2, and Supplementary Figure 1A and C, meta-analysis of MIC values of flavonoids and bitter acids, for 24 hours treatment, resulted in a variety of effectiveness depending on the species. However, in most of the cases flavonoids exert better antimicrobial activity compared to bitter acids when tested in the same bacteria species and strains such as for Staphylococcus aureus (13.61 as opposed to 80.07 μg/mL), Staphylococcus epidermidis (1.37 versus 10.50 μg/mL), Streptococcus salivarius (23.33 versus 58.75 μg/mL) and Streptococcus saprophyticus (2.33 as opposed to 3.83 μg/mL) (Figure 3A). Although antimicrobial tests for 48 hours of treatment were performed in different bacterial species, meta-analysis similarly showed elevated antimicrobial activity of flavonoids compared to bitter acids (Figure 3B, Supplementary Figure 1B and D).

In order to test the antimicrobial effectiveness of various types of hop extracts against microbial species, a meta-analysis was performed with the MIC values for each type of extract testing the effect on each microbial species. As shown in Figure 4A and Table 2, CO₂ (SFE) extracts showed a wide range of activity spanning from being the best antimicrobial agent against Staphylococcus aureus (MIC 6.32 μg/mL) to the worst antimicrobial agent against Streptococcus aureus (MIC 1667 μg/mL). A similar high divergence in antimicrobial activity was shown for CO₂ extracts for 48 hour treatment time and for different microbial species (Figure 4B and Table 2). Hydroalcoholic extracts exposed a more constant antimicrobial activity spanning form 39.0 (Staphylococcus aureus) to 625 μg/mL (Pseudomonas aeruginosa) for 24 hours treatment and from 34.67 (Streptococcus Salivarius) to 384 μg/mL (Candida albicans) for 48 hours treatment.

3.3. Stratification Meta-Analysis of MIC Values of Each Hop Compound

The flavonoids investigated in studies included in the present meta-analysis are xanthohumol (XN) (chalcone) and catechin (flavanol), while bitter acids considered herein refer to humulone (alpha acid) and lupulone (beta acid). Flavonoids and bitter acids belong to different classes of natural compounds, each with distinct biosynthetic origins, structures, and chemical classifications [41,42].

Xanthohumol is a prenylated chalcone characterized by an open-chain flavonoid structure while catechin is a flavan-3-ol with a closed-ring structure and no prenylation. Similarly, humulone and lupulone share a phloroglucinol core but differ structurally: humulone contains an acyl side chain, while lupulone has three prenyl-type side chains, contributing to differences in bitterness and stability [43].

Thus, we stratified our meta-analysis according to each compound to understand the antimicrobial activity of each one, separately. As shown in Figure 5A and Table 3, a meta-analysis was performed with MIC values from experiments with 24 hours of treatment. XN exerted far better inhibition effectiveness against Staphylococcus aureus and Staphylococcus epidermidis (6.53 and 1.37 μg/mL) compared to the alpha acid humulone (83.25 and 18.75 μg/mL), but not compared to beta acid lupulone. Remarkably, against Bacillus Subtilis, Enterococcus faecalis and Streptococcus saprophyticus, XN, humulone and lupulone show similar effectiveness for 24 hours incubation.

Importantly, the other flavonoid catechin, when tested for 48 hours of incubation, revealed a very low antimicrobial activity against Bacillus subtilis and Pseudomonas fluorescens (1200 and 1700 μg/mL, respectively).

Concerning bitter acids, lupulone seems to possess a significantly stronger antimicrobial agent compared to humulone (Figure 5B) for both 24 and 48 hours of treatment.

3.4. Meta-Analysis of MIC Values of Hop Compounds and Extracts for Food Spoilage and Non-Food Spoilage Microorganisms

Given that food spoilage bacteria remain a major concern in food safety and shelf-life extension, and that hop has been used for centuries as a natural preservative in beer, it was intriguing to investigate whether hop compounds exhibit differential antimicrobial activity against food spoilage versus non-food spoilage bacteria. To this end, we stratified our meta-analysis according to the antimicrobial effects of hop-derived compounds and hop extracts against a diverse panel of food spoilage microorganisms, aiming to explore their potential selectivity and application beyond the brewing context [44,45,46].

Μeta-analysis of MIC values for classes of hop compounds (subgrouped according to each compound) and extracts, stratified for food spoilage and non-food spoilage bacteria (Supplementary Table 1), for 24 hours of treatment, showed that XN exerted almost the same effect irrelevant of the food spoilage characteristics of the microorganisms (Table 4 and Figure 6A). Similarly, the α acid humulone showed comparable antimicrobial activities against food spoilage and non-food spoilage microorganism (43.35 and 33.75 μg/mL), while lupulone (beta-acid) showed significantly higher antimicrobial activity against non-food spoilage bacteria (4.20 compared to 12.40 μg/mL, respectively). Importantly, due to the small number of included studies the last results should be considered with caution. For 48 hours of treatment xanthohumol showed enhanced antimicrobial activity against food spoilage compared to non-food spoilage microorganisms (23.46 and 54.32 μg/mL, respectively), as shown in Table 4 and Figure 6B.

Among different types of extracts, hydroalcoholic extracts seemed to be more effective against food spoilage microorganisms for both 24 and 48 hours of treatment. CO₂ extracts were less effective than hydroalcoholic extracts, however, 48 hours treatment resulted in higher antimicrobial effectiveness against non-food spoilage bacteria. Hydralcoholic extracts were more effective against food spoilage bacteria when tested for both 48 hours (Table 4 and Figure 7). MIC values for 48 hours of treatment are expected to be lower than MIC values for 24 hours, because compounds have more time to exert their effect. However, it is important to note here that tests for these different time points are performed with different microorganisms species which profoundly respond in a completely different way (Table 4 and Figure 7).

3.5. Meta-Analysis of MIC Values of Hop Compounds and Extracts for Probiotic and Non-Probiotic Microorganisms

In light of current efforts to develop antimicrobials that are compatible with the human microbiome [47], and the long-standing use of hop compounds as natural antimicrobials, it is compelling to examine whether these compounds can selectively inhibit non-probiotic microorganisms while sparing probiotics. To this end, we conducted a meta-analysis of MIC data from the retrieved studies, to compare antimicrobial activity of hop-derived compounds against probiotic and non-probiotic strains. If such selectivity exists, these compounds could represent high-value functional agents, with potential to support human health by suppressing pathogens while preserving beneficial microbial communities.

To this end the meta-analysis performed showed that antimicrobial activity of XN was stronger against probiotic compared to non-probiotic bacteria (Supplementary Table 1) when incubated for 24 hours (8.49 as opposed to 24.75 μg/mL), (Table 5 and Figure 8A). However this difference was diminished when treatment occurred for 48 hours (Table 5 and Figure 8B). Because there is a significant difference in the number of studies included in each meta-analysis, the last mentioned results should be interpreted with caution (Table 5).

In addition, our meta-analysis revealed that hydroalcoholic extracts were more potent than CO₂ extracts in both 24 and 48 treatment time points (Figure 9A). We should again strengthen the fact that experiments of compounds and extracts performed for different incubation time periods test different microorganisms and this could explain why MIC values in 48 hours are not lower than MIC values in 24 hours.

4. Discussion

The evaluation of the antimicrobial potential of Humulus lupulus (hop) extracts and compounds has consistently highlighted significant variability, arising from differences in bacterial strains, extraction methods, experimental conditions, and the intrinsic diversity of phytoconstituents. The establishment of evidence-based practices, including standardized protocols for assessing antimicrobial activity, is critical for the scientific community to reliably compare, and interpret these findings. Without standardization, evaluating results across studies remains challenging, limiting the translation of hop-derived antimicrobial agents into clinical or commercial applications [48,49].

This systematic review and meta-analysis aimed to combine all available data to deliver a comprehensive, statistically rigorous synthesis of the antimicrobial activity of Humulus lupulus extracts and purified compounds, thereby informing future studies and practical applications. While hops have long been used for their preservative role in brewing, and their pharmacological potential has gained increasing attention, the methodologically diverse nature of existing studies has limited the development of a consolidated view of their antimicrobial efficacy. By integrating MIC data across 450 individual studies and 55 microbial strains, we contribute a much-needed evidence-based framework that enhances the reliability and reproducibility of antimicrobial findings related to hop-derived substances.

Our meta-analysis indicated substantial differences in the antimicrobial efficacy among hop-derived compounds. Specifically, we found pronounced variability in activity between chalcones such as xanthohumol and flavanols like catechin. Xanthohumol consistently showed potent antimicrobial effects, notably against Staphylococcus epidermidis (with MIC value 1.37 μg/mL) and Clostridium species (MIC value 32.6 μg/mL), with significantly lower MIC values compared to catechin, which exhibited minimal activity (MIC value 1200 μg/mL against Bacilus subtilis). Similarly, alpha acids (humulone) demonstrated markedly different antimicrobial profiles compared to beta acids (lupulone), with lupulone generally showing higher potency across multiple microbial species. These findings are in accordance with other reviews highlighting the broad-spectrum antimicrobial and biofilm-inhibiting properties of prenylated chalcones and bitter acids [1,8].

These compound-specific differences underscore the critical importance of chemical structure and functional groups in antimicrobial potency. For instance, the prenylated chalcone structure of xanthohumol, which facilitates cellular membrane penetration, differs substantially from the less effective non-prenylated flavan-3-ol structure of catechin [16,17]. Similarly, structural distinctions between alpha and beta acids - primarily in side-chain composition and prenylation - directly influence their respective antimicrobial activities. Additionally, we observed significant strain-specific variations in antimicrobial susceptibility, highlighting the complexity of microbial responses and reinforcing the need for careful strain selection when evaluating antimicrobial agents.

In addition, hydroalcoholic extracts exhibited more consistent antimicrobial performance than CO₂ extracts, which displayed highly variable outcomes depending on the tested microorganism. In addition, because alpha and beta acids constitute more than 50% of a supercritical fluid-extracted hop extract, its antiproliferative effects are largely driven by bitter acids [27,50]. Given that beer, originally, is the hydroalcoholic solvent for dry hops, data coming from hydroalcoholic extracts are expected to be of more practicability. However, it is also quite a common practice for beer manufacturers to add condense, CO₂-based (SFE) extracts in bulk beer preparations [6] and GmbH & Co. KG. Hops are our passion. BarthHaas. Retrieved May 30, 2025, from https://www.barthhaas.com/. This variability in methodologies adds complexity and discrepancy in research and industrial tests, and underscores the need for careful selection of extraction methods, solvents, and compound standardization - a critical issue raised in other meta-analyses on botanical antimicrobials [53].

An unexpected finding from our study was the variability in antimicrobial activity between the incubation periods of 24 and 48 hours. Contrary to general expectations, many MIC values at 48 hours were higher than at 24 hours. This inconsistency primarily reflects the substantial variation in bacterial species and strains tested across these time points. Similar observations have been reported previously, suggesting differential microbial adaptive responses over extended exposure times [51]. This further emphasizes the urgent need for the scientific community to adopt a standardized panel of microbial strains and time points for evaluating MIC, thus enhancing comparability and reproducibility of antimicrobial studies.

One of the critical contributions of this work is the stratified meta-analysis that distinguishes between effects on food spoilage microorganisms, and probiotics. This functional stratification is of growing importance, especially in the context of microbiome-conscious antimicrobial development. For instance, xanthohumol showed promising selective action, exerting stronger effects on non-probiotic and food spoilage bacteria in several contexts, but also inhibiting probiotics at certain time points - underscoring the complexity of predicting microbial selectivity. Such diversifying behavior reinforces the need for integrated evaluations combining in vitro data with microbiome-sensitive in vivo testing, as proposed in diagnostics-focused meta-evaluations [48,51].

However, our study is not without limitations. Primarily, the absence of complete methodological details in several original studies introduced uncertainties in data interpretation. For instance, variations in experimental protocols such as compound handling, dilution procedures, storage conditions, extraction methodologies, quantitative and qualitative extract composition, bacterial inoculum density, incubation conditions, and solvent composition can significantly influence MIC values. This scarcity of detailed information rendered it impossible to perform meaningful stratification based on these parameters. Additionally, reliance on imputed standard deviations in certain cases could potentially introduce biases into the meta-analysis outcomes. Our analysis also faced methodological challenges due to the inclusion of heterogeneous data, encompassing various hop compounds, extracts, bacterial species, and incubation durations. Despite these difficulties, our rigorous statistical methods - including stratified meta-analyses and sensitivity analyses - allowed us to manage this complexity and derive reliable conclusions. Our findings affirm that hop compounds exhibit reproducible trends in antimicrobial activity, but these can only be reliably interpreted within a unified, evidence-based framework that adequately accounts for methodological heterogeneity. Importantly, the current findings advocate for a more systematic approach to future antimicrobial testing of hop-derived agents. Furthermore, meta-analysis, as demonstrated here and in studies like [48,49,52,53,54] is a powerful tool to extract meaningful trends from heterogeneous datasets, and should be routinely employed in future investigations of natural product pharmacology.

5. Conclusions

In conclusion, this systematic review and meta-analysis confirm the diverse and potent antimicrobial potential of specific hop compounds, notably xanthohumol and lupulone, and underline the significant efficacy of hydroalcoholic extracts against food spoilage and pathogenic bacteria. Significant structural differences among classes of - and individual - compounds necessitate a careful selection and investigation for targeted antimicrobial applications. Nevertheless, variations in methodological approaches and reporting standards continue to obscure definitive assessments of efficacy. To translate the promise of hop-derived compounds into medical, nutritional, or industrial interventions, future research must prioritize methodological standardization, detailed microbial profiling, and integration into microbiome-focused frameworks. Our findings strongly advocate establishing standardized, evidence-based practices in evaluating antimicrobial activity, including uniform microbial panels and consistent experimental protocols. Addressing these methodological gaps will significantly enhance the reliability and comparability of future studies, thus facilitating the broader practical application of hop compounds as valuable antimicrobials in food preservation, pharmaceutical development, and clinical therapeutics. Emphasizing evidence-based approaches will be essential to ensuring the scientific credibility, reproducibility, and practical viability of hop-derived antimicrobials.