Polyphenols in White and Green Shoots of Cultivated Hop (Humulus lupulus L.)

Abstract

Green and white hop shoots are routinely removed during hop production and represent an underutilized biomass stream with potential for valorization. This study evaluated the biomass yield and polyphenolic composition of shoots from three cultivated hop varieties, Aurora, Celeia, and Styrian Wolf. White and green shoots were sampled in 2021, while green shoots were additionally sampled in 2023. Total phenolics were determined by the Folin–Ciocalteu method, and individual polyphenols were quantified by HPLC. Green-shoot biomass removed during training differed significantly among varieties, ranging from 76 kg/ha in Aurora to 188 kg/ha in Styrian Wolf. Total phenolic content of green shoots ranged from 2.22 to 3.47 mg CAE/g dry matter, with Styrian Wolf showing the highest value. Polyphenolic profiles differed markedly between shoot types: white shoots contained higher concentrations of catechin, epicatechin, and naringin, whereas green shoots contained quercetin, higher ferulic acid levels, and the hop-specific prenylflavonoid xanthohumol. Interannual variation was compound-specific at green shoots: the wetter February–April period in 2021 coincided with higher epicatechin and ferulic acid concentrations, whereas naringin and 4-hydroxybenzoic acid were higher in the drier 2023 February–April period. Compared with commonly consumed polyphenol-rich foods, hop shoots contained relatively low concentrations of individual polyphenols; however, their routine generation as an agricultural side stream may make them an attractive raw material for selective recovery of specific compounds, particularly hop-specific xanthohumol.

1. Introduction

Plant polyphenols constitute a diverse class of naturally occurring compounds present in a wide range of fruits, vegetables, teas, and other plant-derived foods. They are well recognized for their antioxidant activity, which contributes to cellular protection against oxidative stress and inflammation and has been associated with reduced risk of several chronic diseases, including cancer, cardiovascular disorders, and Alzheimer’s disease [1,2]. Over 980 naturally occurring antioxidants have been identified; among them, phenolic compounds are dominant. Antioxidants having phenolic groups are also the most widely used ones [3].

Humulus lupulus L., commonly known as hop, is a dioecious perennial climbing plant, primarily cultivated for its female inflorescence, which is rich in alpha-acids and other secondary metabolites. Hop cones are known to contain a broad spectrum of polyphenolic compounds, including phenolic acids, flavonoids, catechins, and hop-specific prenylflavonoids such as xanthohumol [4,5,6]. It is the primary component responsible for imparting flavor, stability, and antimicrobial properties to beer. However, its versatility extends beyond brewing, as they have also found applications in the pharmaceutical and other food sectors. It has been used historically for various medicinal, household, and culinary purposes [7]. Polyphenols in hop cones have been studied for various reasons, especially due to their visible role in industrial applications as natural additives with antimicrobial and/or antioxidant properties [8]. The antioxidant constituents of hop, mainly phenolic compounds, exhibit free radical scavenging properties, as demonstrated by the DPPH assay [9]. Hop cones contain high levels of humulones, lupulones, isohumulones, and xanthohumol, which contribute to their organoleptic properties and have been reported to exhibit antioxidant, anti-inflammatory, antimicrobial, and antitumoral activities [10]. The alpha-acids, beta-acids, and xanthohumol present in this plant’s cones have significant hydroxyl radical scavenging and antioxidant activities [11].

Growing consumer awareness of the links between diet, health, and well-being is increasing demand for foods and ingredients with enhanced nutritional and functional value. In parallel, greater attention to environmental sustainability is stimulating interest in products that are locally sourced, environmentally responsible, and manufactured with a reduced ecological footprint [12]. In this context, underutilized hop tissues and other agro-industrial biomass streams that were previously overlooked are increasingly being investigated as potential sources of antioxidants and bioactive compounds with antibacterial and antiviral properties [13,14].

Given that hop cones are recognized as rich in polyphenolic compounds and that plant by-products are increasingly explored as alternative sources of bioactive constituents, the present study focuses on hop shoots (white shoots and green shoots (see infographics)), which are generated as a routine by-product of hop cultivation and represent an abundant, readily available biomass stream.

In the spring, between 15 to 40 buds emerge on the root system of the cultivated hop. These buds initially grow as white hop shoots underground, remaining white until they are below the surface of the soil. These sprouts are considered to be the first hop plant by-product, as they are mechanically removed during regular pruning of the top of the hop root system in March/April [14]. This agrotechnology measure is performed to promote uniform shoot emergence and subsequent plant development. Pruning removes the previous year’s wood, together with excess buds that have developed on it. This practice serves several important agronomic functions: it regulates the timing and dynamics of hop growth and development; contributes to yield formation by delaying the onset of growth so that flowering coincides more closely with the short-day period, thereby promoting a greater number of inflorescences and potentially higher yield; maintains hop crowns at the appropriate soil depth; and supports the maintenance and monitoring of plant health, particularly with regard to hop downy mildew.

After hop pruning in April, new shoots emerge uniformly from the rhizome. Once they break through the soil surface, they turn green, and when they reach a length of approximately 30 cm, they are considered a delicacy and expensive vegetable according to [15], owing to their very limited availability, which lasts only a few days; however, their flavor is typically more bitter. After boiling, young green shoots have very low lipid content (<0.2 g/100 g), energy value (25 kcal/100 g), and Na content (<40 g/100 g), whereas they are a good source of dietary fiber and vitamins B9 and C [15]. They proved to be a low-fat food (ether extract from 2 to 6% dry matter (DM)) with high protein (from 22 to 30% DM) and fiber content (from 10 to 16% DM) [16]. When they reach a length of 50 to 70 cm, which is typically after three to four weeks of pruning, the initial cleaning and training of the shoots are carried out on the supporting guide, such as twine or string. It is noteworthy that this agrotechnical practice is performed solely by hand and requires individual attention to each plant. In this procedure, six to ten shoots are trained on two to three guides per plant, and one spare shoot is left for possible adjustments later in the season. The remaining shoots are removed and cut away and are typically considered useless plant by-products (waste). Their potential utilization could, however, contribute to circular-economy approaches in hop production by improving resource-use efficiency through the valorization of an otherwise underutilized biomass stream.

This research provides new insights into this waste plant biomass from the perspective of its potential use, thereby also contributing to circular-economy practices. Previous research on cultivated white hop shoots has demonstrated their total phenolic content and antioxidant activity [14]; however, to the best of our knowledge, no studies have characterized the individual polyphenolic compounds present in this plant material. Their identification and quantification were included in the present study. On the other side, individual flavonols have been characterized in young green shoots of wild Humulus lupulus [17]; results showed that hop shoots represent a new source of flavonols. However, profiling of individual phenolic compounds in green shoots of cultivated hop varieties has not yet been reported. The present study therefore aimed to characterize the phenolic composition of this underexplored plant material and, additionally, to quantify the biomass yield of green shoots removed during routine bine training in order to evaluate their potential for valorization as a novel plant-derived resource.

2. Material and Methods

2.1. Location Characteristics

This study was conducted in fully productive hop fields managed by farmers in the traditional hop-growing region of Žalec in the Lower Savinja Valley, Slovenia, on medium deep eutric brown soil on a sandy gravel base. The soil texture of the upper layers was classified as the clay loam to sandy clay loam (i.e., medium-to-heavy soil).

2.2. Plant Material

We collected white and green hop shoots of three distinct varieties, namely Aurora (3300 plants/ha), Celeia (3000 plants/ha), and Styrian Wolf (2800 plants/ha), all bred at the Slovenian Institute of Hop Research and Brewing. Fresh shoot biomass yield per hectare was calculated as the product of the measured biomass per plant and the planting density of the respective plantation.

Medium-early aroma variety Aurora (Northern Brewer × wild male TG growing near the Slovenian Institute of Hop Research and Brewing) and Celeia (tetraploid Savinjski Golding × diploid Yugoslavian male 105/58) are the most widely cultivated hop varieties in Slovenia, accounting each for approximately 40% of the country’s hop fields. Styrian Wolf is a newly introduced variety, boasting very intense aroma and high alpha-acid content (ranging from 13.5 to 18.5% w/w), covering, at the moment, 3% of Slovenia’s hop fields. It is a progeny that resulted from crossing European and American germplasm [18].

2.3. Sampling Methods

In 2021, samples were collected for white and green hop shoots, while in 2023, samples were collected only for green hop shoots, always on the same fields for each investigated variety (

Table 1. Overview of hop shoot samples collected and analyses performed in 2021 and 2023.

Year	Sample Type	No. of Varieties	Replicates	Analyses Performed
2021	White hop shoots	3	3 *	Identification of individual polyphenolic compounds
2021	Green hop shoots	3	3	Total phenolic content; identification of individual polyphenolic compounds
2023	Green hop shoots	3	3	Fresh mass; shoot length; moisture content; dry matter yield total phenolic content; identification of individual polyphenolic compounds;

* One replicate consisted of shoots collected from 40 consecutive plants.

). Both types of shoots were collected on three locations of the same field, each time from 40 consecutive plants. Plants from the edge of the field were omitted. Shoots from each plant were weighted, samples for moisture content were taken, and then shoots from 40 consecutive plants were combined into one sample for chemical analysis (one replication).

White hop shoots were collected just before the routine agrotechnical pruning of hops (in April 2021), when all shoots are mechanically cut back to uniform the plantation, which results in more even plant development across the field and subsequently more uniform flowering and ripening. Soil was manually removed, and shoots were cut from the rootstocks.

Green hop shoots were collected at the time of regular agrotechnical measure training hop shoots (in May 2021 and May 2023). At this agrotechnical measure, workers train 3 to 5 green shoots per twine, which translates to 6 to 10 shoots per plant, while they remove-cut away the remaining shoots (representing waste plant biomass).

In 2021, green hop shoots were analyzed for total phenolic content, while the identification of individual polyphenolic compounds was performed for both white and green shoots. In 2023, green hop shoots were analyzed for total phenolic content, and individual polyphenolic compounds were identified. Additionally, the mass and length of fresh green hop shoots were measured per plant for each investigated variety in this year (before the shoots were combined for chemical analyses); then samples were taken immediately for moisture-content determination and dry matter-yield calculation.

2.4. Data Processing

The data were processed in Microsoft Excel and statistically analyzed using STATGRAPHICS Centurion XVI (StatPoint Technologies, Inc., Warrenton, VA, USA; [19]). The effects of hop variety and production year on the measured parameters were evaluated using a two-way analysis of variance (ANOVA), with both factors treated as fixed effects. The experiment was conducted in three replicates. Prior to ANOVA, the assumptions of normality and homogeneity of variance were assessed. When significant effects were detected, treatment means were compared using Duncan’s multiple range test at a significance level of p ≤ 0.05.

2.5. The Weather Conditions

The weather conditions from October to April—the time after hop harvest and before hop shoot sampling—are presented in Figure 1. In the period from October to December 2020, 381 mm of precipitation was recorded, which is 95 mm above the long-term average. The winter of 2021 was above-average warm, with significant fluctuations in air temperatures, and rich in precipitation. The average daily air temperature values in the first three months in 2021 were higher than the long-term average (1981–2010), and there was 263 mm of precipitation, mainly in the form of rain. After a dry growing season in 2022, the lack of precipitation and above-average temperatures persisted in the last three months of the year. During the period of October to December, only 248 mm of precipitation was recorded. The first three months of 2023 were also warmer than average. In January, there was a significant amount of precipitation (160 mm), but February only had 20 mm. March was relatively dry.

2.6. Chemical Analyses

Moisture content. The moisture content of fresh hop shoots was analyzed using the SIST EN ISO 665:2001 method [20]. To accomplish this, 5 g of each fresh plant sample was weighed into an aluminum pan and subjected to drying at a temperature of 102–104 °C for a duration of 3 h (VO400; Memmert GmbH+Co.KG, Schwabach, Germany). The drying was repeated at least once until constant mass, with all samples analyzed in parallel duplicates.

Total phenolics. Total phenolics were determined by the method of Singleton and Rossi [21]. Five grams of thawed plant material was shredded, and 20 mL of 96% ethanol was added. The solution was extracted for 24 h at 60 °C. After the cooling, the extracts were centrifugated at 3600× g for 10 min. The Folin–Ciocalteu reagent was diluted with deionized water in a 1:1 ratio. Subsequently, 0.5 mL of the diluted Folin–Ciocalteu reagent was added to 1 mL of sample. After an exact interval of 5 min, 0.5 mL of 20% Na₂CO₃ was introduced into the samples. The samples were then left for a period of 90 min at ambient temperature, following which, their absorbance was measured against a blank consisting of 96% ethanol at a wavelength of 746 nm, using a spectrophotometer (UV-1900, Shimadzu, Kyoto, Japan). All supernatants were subjected to analysis in three parallel batches, and the quantification of total phenolics was expressed in units of milligrams of chlorogenic acid equivalents (CAE) per milliliter of extract. To achieve this, a calibration curve was established within the range of 0 to 80 micrograms of chlorogenic acid dissolved in 96% ethanol. The resulting calibration curve was (y = 0.002x − 0.0022), with a high correlation coefficient of R² = 0.998.

Identification of polyphenolic compounds by HPLC. One gram of finely powdered plant material, along with 25 mL of methanol, was ultrasonicated for one hour at 25 °C. Extract solution was filtered through a 0.45 µm PTFE filter into a vial. The analysis was performed using Agilent 1100 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA). For compound separation, a C18 reversed-phase column was used (YMC Triart C18, 150 mm × 4.6 mm, 5 µm). For gradient elution, two mobile phases were applied: A (acetonitrile) and B (5 mM acetic acid). The following program was applied: start with 25% A and 75% B for 10 min, followed by a gradient from 25% A to 100% A over 20 min, and then to 25% A over 10 min. In the next 5 min, the initial conditions were set up. The injection volume was 20 µL, column temperature was 25 °C, and the flow was 1 mL/min. Wavelengths for determination were at 280 nm (catechin, epicatechin, naringin, hydroxycoumarin, and coumarin), 320 nm (caffeic acid, coumaric acid, and ferulic acid), 254 nm (rutin and 4-hydroxybenzic acid), and 370 nm (xanthohumol and quercetin).

3. Results and Discussion

3.1. Total Phenolics in Green Hop Shoots

Research by [14] has demonstrated that the total phenolic content and ferric-reducing antioxidant power of white hop shoots, collected in years 2010 to 2012 from a hop garden in Slovenia, are significantly influenced by hop variety and year, with radical scavenging antioxidant potential varying according to the variety. The total phenolic content, expressed as chlorogenic acid equivalents (CAE) on a dry mass basis, ranged from 0.60 to 1.80 mg/g, and exhibited significant variations across hop variety (cv. Aurora, cv. Celeia, cv. Dana, cv. Hallertauer Magnum and cv. Savinjski golding) and year (2010–2012) (

Table 2. Reported total polyphenol contents in hop (cones, leaves, white shoots, and green shoots) from the literature and this study.

Plant Material	Hop Type/Origin	Variety	Total Polyphenols (Range or Value)	Unit/Basis	Method/Matrix (as Reported)	Source
Hop cones	Cultivated	n.a.	4–14	% DW	Literature range for dried hop cones	[4,5,22]
Hop cones	Cultivated (comparative study across 4 hop-growing regions and 2 years)	Aurora and Hallertauer Magnum	0.80–1.740.74–1.71	mg CAE/mL;	Total polyphenols (Analytica-EBC 9.11, modified); Ethanol extracts of cones	[23]
Hop cones	Cultivated (Czech Rep.)	Multiple	2.21–5.05	mg/g DM	Dried cone material	[24]
Hop cones	Cultivated (Slovakia)	K-72	4.96	mg/g DM	Dried cone material	[25]
Hop leaves	Cultivated; leaves collected after harvest (plant waste); comparative study across 4 hop-growing regions and 2 years	Aurora and Hallertauer Magnum	0.10–0.54 0.19–0.41	mg CAE/mL	Total polyphenols (Analytica-EBC 9.11, modified)	[23]
Hop leaves	Wild hop, leaves harvested in May (Poland)	Wild hop	0.02–6.60	mg GA/g raw material	Methyl, ethyl and isopropyl alcohol extract; ultrasound-assisted extraction	[26]
Hop leaves	Male and female plants of wild hop (Slovakia)	Wild hop	3.27–6.05	mg/g DM	Methanol extracts of leaves	[27]
Hop shoots (white) *	Cultivated (Slovenia)	Multiple	0.60–1.80	mg CAE/g DM;	White (etiolated) shoots, dry mass basis	[14]
Hop shoots (green) **	Cultivated (Slovenia)	Aurora, Celeia, Styrian Wolf	2.2–3.5	mg CAE/g DM;	Green shoots removed during training, dry mass basis	This study

* White hop shoots = etiolated shoots collected prior to routine mechanical pruning. ** Green hop shoots = surplus shoots removed manually during bine training (routine by-product). DW = dry weight; DM = dry matter; CAE = chlorogenic acid equivalents; n.a. = not available. Values are presented as reported in the original sources. Direct comparison across studies is limited due to differences in plant material, extraction solvent (e.g., ethanol vs. methanol), analytical protocols, reporting units (%, mg/g DM, mg/mL extract), and expression basis (e.g., % of dry weight vs. mg CAE/g DM vs. mg CAE/mL extract). “CAE” = chlorogenic acid equivalents; GAE = gallic acid equivalents.

). Although higher values were observed in green shoots in our study, which suggests that shoot developmental stage may contribute to differences in phenolic accumulation, direct attribution to shoot type is not possible, because of methodological and environmental differences between the studies.

In our research, total phenolics content of discarded green hop shoots was analyzed in 2021 and 2023. The results in

Table 3. Effects of hop variety and year on total polyphenol content of green hop shoots, expressed on dry-matter basis. Values are presented as mean ± SD. Variety values are means across 2021 and 2023, whereas year values are means across the three varieties.

Hop Variety	Polyphenol Content (mg CAE/g, Dry-Matter Basis)
Aurora	2.22 ± 0.90 a **
Celeia	2.42 ± 0.98 a
Styrian Wolf	3.47 ± 1.07 b
2021	2.95 ± 0.76 a
2023	2.45 ± 1.34 a

** Different letters within each factor (variety or year) in a column indicate significant differences according to Duncan’s multiple range test (p ≤ 0.05).

show variety- and year-related differences. As the variety × year interaction was not significant, only the main effects of variety and year are presented. Styrian Wolf had the highest polyphenol content (3.47 mg CAE g⁻¹ DM) and differed significantly from cv. Aurora and cv. Celeia (2.22–2.42 mg CAE g⁻¹ DM), which did not differ significantly from each other. At the same time, there were no significant differences between investigated years (2.95 vs. 2.45 mg CAE g⁻¹ DM). However, if we look closer, February–April temperatures were comparable between the two years, whereas precipitation was substantially higher in 2021: 251 vs. 177 mm, approximately 42% more rainfall. Total phenolic content was also higher in 2021, by about 17% relative to 2021 (2.95 vs. 2.45 mg CAE/g DM). The higher total phenolic content recorded in the wetter season may therefore indicate a possible influence of water availability on phenolic accumulation in developing hop shoots. However, because only two growing seasons were investigated, this relationship should be regarded as descriptive rather than causal, and a statistically meaningful correlation between weather variables and phenolic content cannot be established from the present dataset.

Comparing polyphenol content in green hop shoots in our study to hop cones, we can see that there is a moderate phenolic content for this by-product biomass stream. Phenolic levels in hop cones are typically higher; however, reported polyphenol levels in hop cones also vary widely depending on variety, analytical method, and whether total phenols or polyphenols are quantified. For example, total phenols in hop cones have been reported at 11.9–21.2 mg g⁻¹ DM, while Gerhäuser [13] reported that dried hop cones contain polyphenols ranging from 4% to 14%. According to a study by Abram et al. [23], cv. Aurora cones contain approximately 3.9% polyphenols, whereas cones of the cv. Hallertauer Magnum contain 2.0–3.0%. The same study reported that the total phenolics content in the ethanol extracts from cones varied between 0.74 and 1.73 mg CAE/mL and was influenced by factors such as variety, year, and production location. Examination of polyphenols in hop was described by Jelinek et al. [24], who tested the cones of cultivated hop grown in the Czech Republic. Their data show that the amount of polyphenols was between 2.21 mg/g to 5.05 mg/g of DM, which is comparable with the values of polyphenolic substances in the extracts of wild hop cones tested. From a comparison of the data from the study of Urgeova and Polivka [25], where the content of polyphenols in the cultivated variety K-72 grown in Piešťany in 2007 was 4.96 mg/g of DM, it is evident that concentrations of polyphenols in wild hops are similar. The secondary metabolites present in hop cones and their quantities are primarily dependent on the variety, which is related to the plant’s genetic potential for synthesizing specific substances [28,29]. Additionally, factors such as the hop growing area, weather conditions during the growth season [29,30,31], and harvest time [9] also influence the composition of these metabolites in hop cones.

By contrast, hop leaves show lower ranges depending strongly on extraction solvent, year, and whether results are expressed per raw material or per extract (Table 2). Content of polyphenols in leaves of male and female hop wild plants at various localities in Piešťany (Slovakia) was described in the paper of Urgeová et al. [27]; comparison of the results proved that the concentration of secondary metabolites, polyphenols, flavonoids, and bitter acids largely depended on the growing season during the vegetation period. The content of polyphenols (methanol extract) was between 3.27 and 6.05 mg/g DM.

For contextual comparison, green tea leaves generally exhibit substantially higher total phenolic contents than the green hop shoots analyzed in the present study. For example, Murokore et al. [32] reported approximately 9.5–25.8 mg GAE g⁻¹ for green tea, compared with 2.2–3.5 mg CAE g⁻¹ DM in our green hop shoots. Because different extraction procedures and calibration standards were used, these values should not be interpreted as a direct quantitative comparison, but they indicate that hop shoots have a comparatively moderate phenolic density. However, this comparison should be interpreted in the context of biomass availability and use: green tea leaves are a high-value harvested commodity, whereas hop shoots represent a by-product of hop production that is commonly treated as waste or left unused. As another reference, raw pink shallot contains 115 mg total polyphenols per 100 g fresh weight; white common cabbage, 15.30 mg/100 g FW; green lettuce, 65.92 mg/100 g FW; and parsley, 89.27 mg/100 g FW (folin essay; [33]).

3.2. Green Hop Shoots Yield

During bine training, three to five of the most vigorous and uniform shoots are selected and trained onto one support string, preferably those emerging closest to the center of the rootstock. This selection promotes uniform plant development and canopy growth throughout the remainder of the growing season. All remaining shoots are removed; the results indicate that the quantity of them vary substantially among plants and fields, even within the same variety. Cv. Styrian Wolf exhibited the highest weight of waste green shoots per plant (67 g fresh matter, amounting to 188 kg/ha), while the Aurora variety exhibited significantly the lowest values (23 g fresh matter, amounting to 76 kg/ha) (

Table 4. Fresh biomass, shoot length, and moisture content of green hop shoots removed during bine training, by variety, in 2023.

Variety/Genotype	Fresh Biomass (g/Plant; Average)	Fresh Biomass (g/Plant)	Shoot Length (cm)	Moisture (%)
Aurora	23 a *	SD 14; min–max 4–114	23.0 a	81 a
Celeia	51 b	SD 35; min–max 6–183	54.5 c	81 a
Styrian Wolf	67 c	SD 45; min–max 5–211	29.1 b	79 a

* The same letter in a column indicated that there is no significant difference between the values (Duncan test, p = 0.05).

). The within-variety variation in fresh shoot biomass was very high. The coefficients of variation were approximately 61% for Aurora, 69% for Celeia, and 67% for Styrian Wolf. The ranges were wide, especially 4–114 g for Aurora, 6–183 g for Celeia, and 5–211 g for Styrian Wolf. The high variation reflects differences in plant vigor, shoot diameter, position on the rootstock, number of shoots emerging per plant, local soil conditions, and the exact developmental stage at removal.

In the experiment with nine commercial varieties by Ruggery et al. [15] in Italy, the green shoot yield, based on an average of three plants (rootstocks), varied in fresh weight from 15 g per plant of Hallertaurer Aroma (origin New Zealand) in 2014 to 38 g per plant of Cascade variety (origin US) in 2013. The difference with our experiment was that, for shoots in their research, the tops were cut to the marketable length of 20 cm and then weighed. Initially, the shoots ranged from 20 to 40 cm in length. The lower values relative to our study are consistent with their sampling protocol, as restricting measurements to the upper 20 cm inevitably reduces the recorded biomass compared with the full surplus shoots removed during training, which, in our case, also differed in length among varieties.

Also, Rossini et al. [16] reported a significant variety × year interaction (p = 0.0001) for the average fresh weight of green hop shoots per plant in a full productive hop plantation in the Mediterranean environment. Across varieties and years, marketable shoot yield (top 20 cm) ranged from 57 g in Challenger to 205 g in Hallertauer Magnum. All varieties exhibited lower yield in 2018 compared to 2017 (by 50% in Cascade, 38% in Challenger and 62% in H. Magnum).

When comparing our results for green hop shoots with the findings of Vidmar et al. [14] for white hop shoots, it is evident that the biomass of white hop shoots per plant likewise exhibited substantial variability among varieties (3.1–7.1 g dry mass per plant) and was significantly affected by both hop variety and growing year (2009–2011). However, for green hop shoots, the harvested biomass in our experiment was comparable at the Aurora variety and was up to more than twice higher in the case of the Styrian Wolf variety compared to white hop shoots.

When considering the length of the removed shoots, notable disparities were observed. The Celeia variety exhibited significantly the greatest shoot lengths, whereas the Aurora variety displayed significantly the shortest ones. However, this parameter may vary from year to year and among hop fields. Bine training is typically carried out over a period of approximately ten days, determined according to the variety; consequently, shoots in fields trained later within this period may be longer than those in fields where this agrotechnical operation was performed during the first few days. The same is also true for their weight.

3.3. Polyphenolic Compounds in White and Green Hop Shoots

Comparison of polyphenolic profiles revealed clear and statistically significant differences between white and green hop shoots of three Slovenian hop varieties (Aurora, Celeia, and Styrian Wolf), collected from the same fields in the same year (2021). White shoots contained significantly higher concentrations of catechin, epicatechin, and naringin, whereas green shoots were richer in quercetin, ferulic acid, and the hop-specific prenylflavonoid xanthohumol. Rutin content, when averaged across all varieties, did not differ significantly between shoot types; however, variety-specific analysis showed that green shoots of Celeia and Styrian Wolf contained significantly more rutin than their white counterparts, while no difference was observed in Aurora (

Table 5. Polyphenolic composition of white and green shoots of three hop varieties (Aurora, Celeia, and Styrian Wolf) collected in 2021 [µg/g d.m.].

Variety	Shoot Type	Catechin	Epicatechin	Naringin	Rutin	Quercetin	Xanthohumol	Ferulic Acid	4-Hydroxybenzoic Acid
Aurora	white	2.73 ± 0.40 c *	21.33 ± 0.40 d	3.03 ± 0.25 c	0.23 ± 0.06 a	0.00 ± 0.00 a	0.00 ± 0.00 a	0.20 ± 0.00 a	0.17 ± 0.06 bc
Aurora	green	0.83 ± 0.15 a	3.33 ± 0.32 a	0.60 ± 0.10 a	0.20 ± 0.00 a	0.07 ± 0.06 ab	1.73 ± 0.35 c	0.77 ± 0.06 ab	0.10 ± 0.00 ab
Celeia	white	1.87 ± 0.12 b	13.93 ± 2.02 b	2.13 ± 0.40 b	0.23 ± 0.06 a	0.00 ± 0.00 a	0.00 ± 0.00 a	0.17 ± 0.06 a	0.13 ± 0.06 abc
Celeia	green	1.10 ± 0.35 ab	2.87 ± 0.47 a	0.67 ± 0.25 a	0.40 ± 0.10 b	0.03 ± 0.06 ab	1.27 ± 0.35 b	1.13 ± 0.38 b	0.10 ± 0.00 ab
S. Wolf	white	1.20 ± 0.53 ab	16.03 ± 1.27 c	3.73 ± 0.35 d	0.30 ± 0.00 ab	0.00 ± 0.00 a	0.00 ± 0.00 a	0.17 ± 0.06 a	0.20 ± 0.00 c
S. Wolf	green	1.70 ± 0.50 b	3.03 ± 0.23 a	0.70 ± 0.10 a	0.40 ± 0.10 b	0.10 ± 0.00 b	1.13 ± 0.25 b	6.33 ± 0.98 c	0.07 ± 0.06 a
ANOVA p		0.00	0.00	0.00	0.01	0.01	0.00	0.00	0.02

* Values are presented as mean ± SD (n = 3). The same letter in the column indicates that there is no significant difference between treatments (Duncan multiple test, p ≤ 0.05).

). These findings underline the importance of considering varietal interactions when assessing the phenolic composition of hop shoots.

In 2021, among the quantified compounds, epicatechin was the most abundant polyphenol in both white and green shoots. Its concentrations were markedly higher in white shoots, particularly in the Aurora variety, where white shoots reached 21.33 µg/g d.m. (Table 5). Catechin levels were consistently lower than those of epicatechin across all samples. Epicatechin is a widely distributed flavonoid in plant-derived foods such as apples, berries, cocoa, and tea, where it commonly occurs at concentrations in the mg/g fresh-weight range [33,34]. Compared with these dietary sources, epicatechin levels in hop shoots were several orders of magnitude lower, indicating that hop shoots cannot be considered a nutritionally relevant source of this compound through direct consumption.

Naringin, a flavanone glycoside best known from citrus fruits, was present at significantly higher levels in white shoots than in green shoots, with the highest concentration detected in white shoots of Styrian Wolf (3.73 µg/g d.m.). Nevertheless, naringin concentrations in hop shoots were extremely low compared with grapefruit juice and other citrus products, where contents commonly reach tens of mg per 100 g or 100 mL [33,35]. White hop shoots may therefore represent only a marginal source of naringin.

Trans-ferulic acid was significantly more abundant in green hop shoots, particularly in the Styrian Wolf variety, where concentrations reached 6.33 µg/g d.m. Ferulic acid is a ubiquitous phenolic acid in plant cell walls, often present at g/kg levels in cereal brans and other agricultural by-products. In comparison with such materials, hop shoots again contained relatively low amounts, although the preferential accumulation in green tissue suggests that shoot developmental stage influences phenolic acid metabolism.

Xanthohumol, a prenylated flavonoid considered characteristic of hops, was detected exclusively in green shoots, with concentrations ranging from 1.13 to 1.73 µg/g d.m. depending on variety. Its absence from white shoots and higher abundance in green shoots is consistent with its biosynthesis in photosynthetically active tissues and supports the notion that green hop shoots represent a more suitable raw material when targeting hop-specific prenylflavonoids. Although absolute concentrations were low, the large biomass of shoots removed during routine hop cultivation suggests that green shoots may still be of interest for targeted extraction approaches.

4-Hydroxybenzoic acid concentrations showed a more complex pattern. When averaged across varieties, white shoots contained significantly higher levels than green shoots (Table 5). However, variety-specific data revealed higher concentrations in green shoots of Celeia and Styrian Wolf (Table 5), indicating that both shoot type and genotype influence accumulation of this compound. Absolute levels remained very low compared with common dietary sources, such as berries [33].

Quercetin was detected only at trace levels in green shoots and was not detected in white shoots. These results contrast with findings reported for wild hop shoots from Northern Italy, where flavonols—particularly kaempferol derivatives and quercetin glycosides—were among the dominant phenolics [17]. The discrepancy is likely attributable to differences in genotype (wild vs. cultivated hops), environmental conditions, and the analytical focus on aglycones rather than conjugated flavonol forms.

The strongest varietal effects in 2023 (

Table 6. Quantification of polyphenols in green hop shoots in various hop varieties (Aurora, Celeia, and Styrian Wolf) in 2023 [µg/g d.m.].

Variety	Shoots Type	Catechin	Epicatechin	Naringin	Rutin	Quercetin	Xanthohumol	Ferulic Acid	4-Hydroxybenzoic Acid	Caffeic Acid	p-Coumaric Acid
Aurora	green	0.50 ± 0.33 a *	0.59 ± 0.32 a	1.48 ± 0.44 b	0.41 ± 0.19 a	0.040 ± 0.000 a	1.81 ± 0.43 a	0.10 ± 0.06 a	0.17 ± 0.02 a	0.16 ± 0.06 a	0.083 ± 0.015 a
Celeia	green	1.27 ± 0.21 b	1.71 ± 0.80 a	1.48 ± 0.44 b	0.49 ± 0.27 a	0.083 ± 0.021 b	1.02 ± 0.29 a	0.42 ± 0.20 b	0.20 ± 0.08 a	0.26 ± 0.10 a	0.17 ± 0.06 b
S. Wolf	green	1.64 ± 0.44 b	0.91 ± 0.23 a	1.74 ± 0.16 b	0.47 ± 0.19 a	0.067 ± 0.006 b	1.46 ± 0.41 a	0.84 ± 0.10 c	0.24 ± 0.03 a	0.39 ± 0.13 a	0.37 ± 0.03 c
ANOVA p		0.02	0.09	0.03	0.88	0.01	0.11	0.00	0.34	0.08	0.00

Values are presented as mean ± SD (n = 3). * Different letters within a column indicate significant differences among varieties according to Duncan’s multiple range test (p ≤ 0.05). Coumarin and hydroxycoumarin were not detected (ND) in any sample and were therefore excluded from the statistical analysis.

) were found for ferulic acid and p-coumaric acid, with Styrian Wolf showing significantly higher concentrations than both Celeia and Aurora. Significant varietal differences were also observed for catechin, naringin, and quercetin. In contrast, epicatechin, rutin, xanthohumol, 4-hydroxybenzoic acid, and caffeic acid did not differ significantly among varieties. Because n = 3 per variety is rather small, we should also notice marginal differences at caffeic acid (p = 0.082) and epicatechin (p = 0.09).

The general polyphenolic profile of green hop shoots was broadly maintained between 2021 and 2023, but the concentrations of several individual compounds varied considerably, indicating that the year effect was compound-specific rather than uniform across the phenolic profile. The most pronounced interannual differences were observed for epicatechin, naringin, ferulic acid, and 4-hydroxybenzoic acid. Epicatechin was lower consistently across all three varieties, with an average of approximately 3.08 µg/g DM in 2021 and 1.07 µg/g DM in 2023, corresponding to a difference of about 65%. Ferulic acid showed even much lower value, in an average of approximately 83%; in Styrian Wolf, for example, its concentration was 6.33 and 0.84 µg/g DM, respectively. In contrast, naringin was consistently higher in 2023, by approximately 107% on average, while 4-hydroxybenzoic acid was also higher in all three varieties. Rutin tended to be higher in 2023, whereas catechin and xanthohumol remained comparatively stable overall, and quercetin showed no consistent interannual trend.

Some variety-specific characteristics were nevertheless maintained across years. Styrian Wolf, for example, had the highest ferulic acid concentration in both seasons despite the substantially lower absolute values in 2023, and it remained among the varieties with relatively high catechin and naringin contents. These results therefore suggest that both genotype and growing season contribute to the phenolic composition of green hop shoots. Mean air temperature during February–April was nearly identical in 2021 and 2023 (6.7 and 6.6 °C, respectively), whereas cumulative precipitation differed markedly, reaching 251 mm in 2021 compared with 177 mm in 2023. The contrasting precipitation regimes may therefore have contributed to the observed interannual variation in individual phenolic compounds. However, because only two growing seasons were investigated, the influence of precipitation cannot be separated from other year-specific environmental and developmental factors, and the relationship should be interpreted as descriptive rather than causal. This interpretation is consistent with previous reports showing that phenolic composition in hop cones can vary substantially among growing seasons and in response to climatic and cultivation conditions [36].

Comparison with selected commonly consumed vegetables, herbs, and fruits (

Table 7. Comparison of selected individual polyphenols in hop shoots and representative plant-derived foods.

Polyphenol (Compound)	Hop Shoots—White (µg/g d.m.; ≈µg/g Fresh Shoots) *	Hop Shoots—Green (µg/g d.m.; ≈µg/g Fresh Shoots) *	Example Vegetable, Herb, Fruit (Typical Content) mg per 100 g Fresh Weight Unless Otherwise Mentioned **
(+)-Catechin	1.93; ≈0.33	1.21; ≈0.24	Green bean, raw: 0.41
(−)-Epicatechin	17.10; ≈2.91	3.08; ≈0.62	Green bean, raw: 0.69 Blackberry, raw: 11.48
Naringin	2.97; ≈0.50	0.66; ≈0.13	Rosemary, fresh: 55.1
Rutin (quercetin-3-O-rutinoside)	0.26; ≈0.044	0.33; ≈0.067	Asparagus, raw: 23.20 Salad (green, raw): 0.04 Green bean, raw: 0.13
Quercetin	0.00; ≈0.00	0.07; ≈0.014	Onion [Red], raw: 1.31 Black elderberry: 42.0
Ferulic acid	0.18; ≈0.031	2.74; ≈0.553	Cauliflower, raw: 0.53
4-Hydroxybenzoic acid	0.17; ≈0.029	0.09; ≈0.018	American cranberry: 0.42 Carrot, raw: 0.05 Olive [Green], raw: 4.97

* Fresh-weight conversion for hop shoots: using our moisture contents (white, 83.0%; green, 79.8%), converted as follows: µg/g FW ≈ (µg/g d.m.) × dry-matter fraction (0.17 for white; 0.202 for green). ** Analytical method reported for the cited food data: LC chromatography (HPLC/UPLC) values collated in Phenol-Explorer, version 6.3.

) indicates that hop shoots generally contain relatively low concentrations of the individual polyphenols quantified in this study. Accordingly, hop shoots should not be regarded as particularly rich dietary sources of these compounds. Their potential value lies instead in their use as a seasonal niche vegetable and, importantly, as an annually generated and largely underutilized side stream of hop production that could be valorized within circular-bioeconomy approaches. Considering the relatively low concentrations of individual phenolics in fresh shoots and the potential sensory limitations associated with bitterness, targeted recovery of selected compounds, particularly hop-specific constituents such as xanthohumol from green shoots, may represent an attractive alternative valorization pathway alongside direct food use.

4. Conclusions

Green hop shoots contained moderate levels of total phenolics, with significant varietal differences and the highest values observed in Styrian Wolf. Although mean total phenolic content was higher in 2021 than in 2023, the year effect was not statistically significant. The higher precipitation recorded during February–April 2021 coincided with higher phenolic levels, suggesting that water availability may contribute to interannual variation; however, this relationship remains descriptive because only two growing seasons were evaluated.

Compared with other hop tissues and phenolic-rich plant materials, green hop shoots appear to represent an intermediate source of phenolics. Their main relevance therefore lies not in exceptionally high phenolic content, but in their availability as an annually generated and largely underutilized side stream of hop production, supporting their potential valorization as a raw material for phenolic extraction and circular-bioeconomy applications.

The results demonstrate that routine bine training generates a substantial and predictable side stream of fresh plant material, particularly in variety Styrian Wolf, supporting further evaluation of green hop shoots as a potentially exploitable raw material. The very high within-variety variability indicates that shoot biomass is strongly influenced by plant vigor and by the exact time at which training is performed.

Shoot length showed a similarly strong varietal effect, but this trait should also be interpreted cautiously because training is conducted over a variety-dependent period of approximately ten days, during which both shoot length and biomass may continue to increase. For agronomic assessment and valorization planning, biomass expressed per hectare therefore provides a more meaningful indicator than shoot length or biomass per plant alone.

Hop shoot polyphenolic composition was strongly shaped by shoot type, variety, and growing year: white shoots were richer in catechin, epicatechin, and naringin, whereas green shoots contained more ferulic acid and quercetin and uniquely contained the hop-specific prenylflavonoid xanthohumol. Although concentrations of individual compounds were generally low compared with conventional dietary plant sources, the distinct phenolic profile of hop shoots—together with their annual generation as an underutilized side stream—supports their potential valorization, particularly through targeted recovery of selected bioactive compounds and broader circular-bioeconomy applications in hop production.