The Influence of Plant Growth Regulators (PGRs) on Physical and Chemical Characteristics of Hops (Humulus lupulus L.)

Abstract

Hops (Humulus lupulus L.) are a critical component in beer brewing. The growing demand for craft beer has increased interest in hop cultivation in non-traditional regions where unfavorable climatic conditions hinder optimal yield and quality. To address these challenges, this study investigates the effects of plant growth regulators (PGRs) on hop cone yield and chemical compositions. In two separate studies, year-1 Cascade hops were subjected to various PGR treatments in the field. PGR treatments generally had minimal effect on the dry cone yield in study I. In study II, a combination of Ethephon at 45 mg/L and ProGibb at 3 mg/L significantly increased the cone yield by 125% compared to the control. While all treatments had a “good quality” hop storage index, a combination of Ethephon and ProGibb produced alpha acid percentages within the commercial standard range. Ethephon at 30 mg/L combined with ProGibb at 2 mg/L enhanced bitterness and aroma, delivering the highest concentration of volatile organic compounds at 569.7 mg/L, thereby enhancing aroma compounds associated with fruity esters, monoterpenes, and sesquiterpenes. This study demonstrates that specific PGR treatments can improve the chemical composition of hops grown in non-traditional regions, with implications for optimizing aroma and bitterness in beer.

1. Introduction

Hops (Humulus lupulus L.) are an essential ingredient in brewing beer, with the lupulin glands in female hop cones producing a resin rich in organic acids and essential oils. These compounds contribute to the bitterness, flavor, and preservation of beer [1,2,3,4]. While about 98% of hops are used in brewing, research also highlights their potential medicinal benefits, such as alleviating symptoms of mental stress, insomnia, and other medical conditions [5,6,7,8]. Beyond brewing, hop oils and extracts are increasingly utilized as flavoring and aromatic additives in various products, including flavored sodas (Hop), hair styling products [9], balms [10], flavored waters [11], and liquors [12].

Hops are one of the four key ingredients specified by the Reinheitsgebot, the German beer purity law. They contribute bitterness, aroma, flavor, stabilizing foam, and protection against Gram-positive bacteria in beer [13]. When added during the boil, hops impart bitterness and some pleasant aromas; however, most aromatic compounds tend to dissipate during this process [13]. The bitterness derived from hops is primarily associated with hop resin, which can be categorized into hard and soft resins. Brewers focus mainly on the soft resin, composed of alpha and beta acids. The primary components of alpha acids include humulone, cohumulone, and adhumulone, which undergo isomerization during boiling to create the bitterness characteristic of “bitter” beers. In contrast, beta acids are less significant in brewing because they are less soluble and do not isomerize during boiling [13]. While hops are essential for imparting bitterness, their most significant contribution lies in their aromatic qualities. The impact of hops on the aromatic composition of beer has been extensively studied over the years. Researchers have focused on identifying key compounds, particularly those found in the extracted oil phase of hops [14,15,16,17,18]. Additionally, other studies have explored how the loss of hop volatiles during brewing affects beer flavor [19,20,21,22]. This extensive research has led to a consensus on the chemical compounds that play a crucial role in the flavor and aromatic profile of beer.

As sales of craft beer products in the U.S. continue to soar [23], the demand for hops has risen accordingly [24]. In response to the rapid growth and diversification of the craft beer industry, hop cultivation in the southern U.S. has increased significantly, driven by the high prices of hops and the desire to produce locally sourced ingredients [23]. The demand for local, flavorful beer brewed with locally grown hops is robust [25]. However, due to climate limitations, hop yields remain low in southern regions, such as Florida, making current production levels economically unprofitable.

As a temperate crop, hops thrive under long days (more than 14 h of daylight) and typically require an extended period of low temperatures to achieve high economic yields [26,27]. Although hops have been cultivated for hundreds of years in the U.S., production is mainly concentrated in regions like the Pacific Northwest, where summers are warm with long days and winters provide cool, long nights. In contrast, the southern U.S. lacks the necessary day length and chilling hours that favor optimal hop growth, making it more challenging for local growers to achieve high productivity and profitability [26]. Traditionally, it has been believed that hops require approximately six weeks of chilling temperatures between 2 and 5 °C to promote uniform cone flowering. Insufficient chilling hours were thought to result in premature reproductive transition and excessive vegetative growth, hindering flowering and cone production. However, recent research by Bauerle [28] suggests that vernalization and dormancy do not significantly affect hop flowering or cone yield, opening new possibilities for hop production in the southern U.S. Consequently, developing economically viable and practical strategies to overcome climate-related challenges is crucial for improving hop yields and making cultivation profitable for local growers, helping to meet the demands of the region’s craft beer industry.

Plant growth regulators (PGRs) are commonly used in horticulture to manage various aspects of plant development, including controlling or promoting growth, increasing crop biomass and yield, breaking dormancy, encouraging rooting, improving branching, and enhancing stress tolerance [29,30]. The effectiveness of PGRs can be influenced by several factors, including the active ingredients used, crop species, timing and method of application, application concentration, and environmental conditions [30,31]. Common PGRs include auxins [such as indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA)], gibberellin acids (GA), anti-gibberellins (such as chlormequat chloride), ethylene-generating compounds (such as Ethephon), and cytokinins [such as 6-benzylaminopurine (BA)] [32]. Research has shown that GA can typically increase stem elongation in cut flowers [33] and break bud dormancy [34]. Chlormequat chloride is often used to promote flowering and suppress extension growth [35]. Ethephon has been found to reduce apical dominance, thereby increasing branch formation, flowering, and overall yield [32]. 6-BA is typically used to enhance lateral branching [36] and promote flower bud formation [37]. These diverse active ingredients highlight the versatility of PGRs in optimizing plant growth and productivity across various horticultural practices.

Despite the long history of hop cultivation, research on the effects of PGRs on hop growth and development remains limited. While most research focused on the effects of PGRs on cone yield and bittering acids, little attention has been given to their influence on volatile compounds that affect aroma. For example, Zimmermann et al. [38] found that 5 mg/L of GA₃ applied to hop plants at the 1.5 m height stage significantly increased cone yield by 340 kg per ha over a three-year period. However, GA₃ applications led to smaller and more brittle cones when applied at flower set. In a separate study, Thomas and Goldwin [39] indicated that GA₃ applied prior to hop flowering increased the yield of some hop cultivars by increasing cone numbers without changes in alpha-acid content. The most recent study on hops tested four PGRs on greenhouse-grown hops [40]. However, only one PGR, Sumagic (active ingredient: uniconazole), was further evaluated for its effects on cone yield. To bridge the gap, the objective of our study was to investigate the effects of different concentrations and active ingredients of PGR treatments on cone yield, acid concentration, and Volatile Organic Compound (VOC) composition in hops. This study aims to provide empirical insights into optimizing hop cultivation to produce commercially acceptable hops in challenging climatic conditions to enhance the economic viability of hop production in non-traditional growing regions.

2. Materials and Methods

2.1. Study I—The Effect of PGR Active Ingredients and Concentrations on Hop Cone Yield

2.1.1. Plant Material, Experiment Site, and Management

The hop cultivar ‘Cascade’ was selected based on its popularity and demand by craft brewers and its good growth characteristics when cultivated in Florida [41]. Rhizomes were sourced from a commercial nursery, Great Lakes Hops (Zeeland, MI, USA), and initially acclimated in a polycarbonate greenhouse until they were ready for transplanting.

A field experiment was conducted at the University of Florida Mid-Florida Research and Education Center (Apopka, FL, USA; 28.64° N, 81.55° W) and was originally planned for two consecutive years. However, due to the impact of COVID-19 in 2020, the study was only conducted in 2019. The tall-trellis system used in this study was described in detail by Pearson and Smith [42]. In brief, the trellis was constructed using 6 m poles and galvanized cables. Each plant was supported by two coir fiber twines running from the ground to the overhead cable, with three to four bines trained clockwise onto each twine. Pine straw mulch was also applied to suppress weed growth.

A total of 117 plants were transplanted in rows on 1 May 2019 and were subjected to natural day length. The plants received daily irrigation through micro-irrigation emitters, supplemented with a soluble fertilizer (Peters Excel pHLow; ICL Specialty Fertilizers, Summerville, SC, USA). The soluble fertilizer contained 18% nitrogen (N), 8% phosphorus (P), and 15% potassium (K). Plants received 70 mg/L N until mature (3.5 months post-transplant). Mature plants were supplied with 150 mg/L N.

2.1.2. PGR Treatments

Four PGRs, including ProGibb LV Plus (Valent BioSciences Corporation, Libertyville, IL, USA) (hereafter referred to as ProGibb), MaxCel (Valent BioSciences Corporation, Libertyville, IL, USA), Cycocel (OHP, Inc., Mainland, PA, USA), and Ethephon 6 (RedEagle International, LLC., Lakeland, FL, USA) (hereafter referred to as Ethephon), were used. The hop plants were divided into 39 blocks, each containing three plants. The PGR treatment was randomly assigned to each block, with three replications per treatment. The application rate and application method were determined based on the label recommendation. PGR treatments were applied when hop bines were approximately 0.3–1.2 m or 1.5–2.4 m in height to evaluate the influence of PGR application timing on hop plant maturity and subsequent cone development and yield. A detailed description of the PGR treatment, including the active ingredient, application rate, method, and timing, is provided in

Table 1. Types of plant growth regulators, application rates, methods, and timings applied to the ‘Cascade’ hops in Study I.

Treatment	Name	Active Ingredient	Application Rate	Application Method	Bine Height When Applied (m)
1	Control (Water)	NA	NA	Spray	1.5–2.4
2	ProGibb	Gibberellic acid	4 mg/L (ppm)	Spray	0.3–1.2
3	ProGibb	Gibberellic acid	4 mg/L (ppm)	Spray	1.5–2.4
4	ProGibb	Gibberellic acid	7 mg/L (ppm)	Spray	0.3–1.2
5	ProGibb	Gibberellic acid	7 mg/L (ppm)	Spray	1.5–2.4
6	MaxCel	6-Benzyladenine	50 mg/L (ppm)	Spray	1.5–2.4
7	MaxCel	6-Benzyladenine	250 mg/L (ppm)	Spray	1.5–2.4
8	Cycocel	Chlormequat-chloride	600 mg/L (ppm)	Drench	1.5–2.4
9	Cycocel	Chlormequat-chloride	1200 mg/L (ppm)	Drench	1.5–2.4
10	Ethephon	Ethephon phosphonic acid	50 mg/L (ppm)	Spray	1.5–2.4
11	Ethephon	Ethephon phosphonic acid	200 mg/L (ppm)	Spray	1.5–2.4
12	Cycocel + Ethephon	Chlormequat-chloride + Ethephon phosphonic acid	600 + 50 mg/L (ppm)	Drench + Spray	1.5–2.4
13	Cycocel + Ethephon	Chlormequat-chloride + Ethephon phosphonic acid	1200 + 200 mg/L (ppm)	Drench + Spray	1.5–2.4

NA = not applicable.

.

2.1.3. Hops Harvest and Data Collection

Hop cones were harvested when mature, with the initial harvest on 27 June 2019. Immediately after harvest, samples were dried at 51 °C for 24 h using a lab oven (Model 40 GC, Burr Ridge, IL, USA) to a moisture content of approximately 8–10% (w/w). A summation of dry cone yield was calculated at the end of the study.

2.2. Study II—The Effect of Gibberellic Acid and Ethephon on Hop Growth, Yield, Acid Concentration, and VOC Composition

The plant materials, experimental site, and management practices were similar to those in Study I, except for the hop transplanting date on 22 April 2021.

2.2.1. PGR Treatments

In Year 2021, two PGRs, ProGibb and Ethephon, were further evaluated at lower concentrations on newly planted hops. These treatments were selected based on the cone yield results from Study I. The PGR treatments were applied as a foliar spray once, targeting early bines when the plants were approximately 0.3–1.2 m tall.

Table 2. Plant growth regulators, active ingredients, and application rates applied to the ‘Cascade’ hops in Study II.

Treatment	Name	Active Ingredient	Application Rate
1	Control (Water)	NA	NA
2	Ethephon	Ethephon phosphonic acid	15 mg/L (ppm)
3	Ethephon	Ethephon phosphonic acid	30 mg/L (ppm)
4	Ethephon	Ethephon phosphonic acid	45 mg/L (ppm)
5	Ethephon	Ethephon phosphonic acid	60 mg/L (ppm)
6	ProGibb	Gibberellic acid	1 mg/L (ppm)
7	ProGibb	Gibberellic acid	2 mg/L (ppm)
8	ProGibb	Gibberellic acid	3 mg/L (ppm)
9	ProGibb	Gibberellic acid	4 mg/L (ppm)
10	Ethephon + ProGibb	Ethephon phosphonic acid + Gibberellic acid	15 + 1 mg/L (ppm)
11	Ethephon + ProGibb	Ethephon phosphonic acid + Gibberellic acid	30 + 2 mg/L (ppm)
12	Ethephon + ProGibb	Ethephon phosphonic acid + Gibberellic acid	45 + 3 mg/L (ppm)
13	Ethephon + ProGibb	Ethephon phosphonic acid + Gibberellic acid	60 + 4 mg/L (ppm)

NA = not applicable.

provides a detailed description of the PGR treatments, including the active ingredient and application rate.

2.2.2. Hops Harvest and Data Collection

Similar to Study I, hop cones were harvested when mature, with cone yield summed at the end of the experiment. Samples were dried at 51 °C for 24 h using a lab oven (Model 40 GC, Burr Ridge, IL, USA) immediately after harvest to a moisture content of approximately 8–10% (w/w). Samples were vacuum-sealed in clear plastic bags and stored at 4 °C until further chemical analysis could be conducted.

2.2.3. Alpha and Beta Acids

Alpha and beta acids were analyzed using the American Society of Brewing Chemists (ASBC) official method, Hops-6 [43].

α-Acids % = d * (−51.56A₃₅₅ + 73.79A₃₂₅ − 5.10A₂₇₅)

(1)

β-acids % = d * (55.57A₃₅₅ − 47.59A₃₂₅ + 5.101A₂₇₅)

(2)

d is the dilution factor, and A₃₅₅, A₃₂₅, and A₂₇₅ are the absorbances of the wavelengths at which the diluted sample was measured.

2.2.4. Hop Storage Index (HSI)

The HSI was calculated following the method described in the ASBC official method Hops-6 [43].

HSI = A₂₇₅/A₃₂₅

(3)

A₃₂₅ and A₂₇₅ are absorbances for the wavelengths at which the diluted sample was measured.

2.2.5. Essential Oil of Volatile Compounds

Essential Oil Extraction

The essential oil was extracted from Florida-grown hops using the ASBC official method Hops-13 with modifications. Approximately 110–120 g of frozen vacuum-sealed hops were weighed and added directly into a 20 L evaporation flask (Büchi, Flawil, Switzerland) with 2 L of deionized (DI) water. The hops were ground in the flask using a Waring Commercial 0.4 m Heavy-Duty Immersion Blender (Waring, Stamford, CT, USA) for one minute, followed by adding 1 L of DI water. The evaporation flask was connected to a Büchi R-220 SE Rotavapor (New Castle, DE, USA) to perform vacuum distillation. Vacuum distillation was chosen for VOC characterization in hop oil to capture and preserve the volatile compounds without severe temperature degradation. The vacuum pressure was set to 115 mBar, resulting in the boiling point of the ground hop and water mixture being ~49 °C. The evaporation flask was set to 30–50 RPM to induce optimal heat transfer and uniform mixing. The water bath was initially set to 69 °C for ~2–2.5 h, then increased to 90 °C for ~2 h until 90% or greater of the water was recovered. The distilled water and hop oil mixture was drained from the receiving flask into a 4 L Erlenmeyer flask.

A Folch extraction of the lipid portion of the mixture was performed using a 2:1 chloroform to methanol mixture at a volume of 50 mL in the Erlenmeyer flask. The lipid/chloroform/water mixture was transferred into a 500 mL separatory funnel with an approximate volume of 400–450 mL. 5% NaCl salt solution was added at a volume of 5 mL to the separatory funnel to remove residual water from the lipid portion. 10 mL of the lipid portion was fractionated into Gas Chromatograph (GC) glass sample vials with screw caps (Thermo Fisher Scientific, Waltham, MA, USA). 2-octanol was added as an internal standard at a concentration of 1 µL (81.9 mg/L) in 10 mL of lipid. The hop oil/lipid sample was injected within 24 h.

Gas Chromatography—Mass Spectrometer (GC-MS) Analysis of the Essential Oil

VOCs were analyzed by injecting 10 uL of sample into the injection port of a Shimadzu GC-2010 Plus Series mass spectrometer detector (MSD) QP2010 SE (Columbus, MD, USA). The injection port was set to 250 °C, and all injections were made in split mode using a 15:1 ratio with a narrow-bore, deactivated glass insert. A 4.5-min solution delay was utilized to protect the detector. VOCs were separated using a nonpolar ZB-5MS (ZB; 30 m * 0.25 mm id * 0.25 μm film thickness) with helium as the carrier gas at a flow rate of 2.0 mL/min (linear velocity 53.8 cm/s). The GC oven temperature program was 35 °C, held for 5 min, and then increased to 225 °C at a rate of 6 °C/min. Once the final temperature of 225 °C was reached, it was maintained for 10 min. The MSD was maintained at 200 °C, and sample mass was scanned in the 40–800 m/z range. GC-MS was performed to identify the volatile and semi-volatile compounds present in the samples. Peaks were identified using standardized retention time (retention index values, RI), pure compounds, and fragmentation spectra of standards, and the Wiley 2014 mass spectral library [44].

Identification

Volatile compounds were identified based on their LRI (linear retention index) values using nonpolar (ZB-5MS) columns (30 m × 0.25 mm i.d., 0.25 μm film; J&W, Folsom, CA, USA). The RI values were compared to literature values. Aliphatic hydrocarbon standards were analyzed in the same manner using a ZB-5MS column to calculate RI:

RI = 100N + 100n (t_Ra − t_Rn)/(t_R(N+n) − t_RN)

(4)

N is the carbon number of the lowest alkane, and n is the difference between the carbon number of the two n-alkanes that are bracketed between the compound; t_Ra, t_Rn, and t_R(N+n) are the retention times of the unknown compound, the lower alkane, and the upper alkane, respectively [44].

Compound Response

The GC-MS peak area of each identified compound was normalized against the peak area of the internal standard 2-Octanol in each chromatogram. This relative response was compiled for each compound and used in statistical analysis.

2.3. Plant Growth Environmental Conditions

Environmental conditions in the hop field for Studies I and II were monitored using data from the Florida Automated Weather Network (FAWN).

Table 3. Means (±SD) of average air temperature at 60 cm, 2 m, and 10 m above ground, soil temperature at −10 cm, relative humidity (RH), and solar radiation at 2 m, and wind speed at 10 m above ground measured by Florida Automated Weather Network (FAWN).

		Air Temperature (°C, 60 cm)	Air Temperature (°C, 2 m)	Air Temperature (°C, 10 m)	Soil Temperature (°C, −10 cm)	RH (%, 2 m)	Solar Radiation (w/m2, 2 m)	Wind Speed (mph, 10 m)
Study I	May 2019	26.1 ± 0.3	25.9 ± 0.3	25.6 ± 0.3	30.1 ± 0.4	73.2 ± 1.3	238.2 ± 10.7	5.5 ± 0.2
	June 2019	27.3 ± 0.3	27.1 ± 0.3	26.7 ± 0.3	30.4 ± 0.4	81.6 ± 1.3	205.7 ± 9.7	5.2 ± 0.2
	July 2019	27.2 ± 0.2	26.9 ± 0.2	26.6 ± 0.2	30.9 ± 0.2	83.8 ± 0.9	195.8 ± 10.4	4.4 ± 0.2
	August 2019	27.7 ± 0.2	27.5 ± 0.2	27.0 ± 0.2	30.9 ± 0.2	84.8 ± 0.8	234.2 ± 10.4	4.6 ± 0.2
Study II	May 2021	25.2 ± 0.3	24.9 ± 0.3	24.5 ± 0.3	30.1 ± 0.2	67.9 ± 1.4	286.1 ± 8.5	6.4 ± 0.3
	June 2021	27.0 ± 0.3	26.6 ± 0.3	26.2 ± 0.3	30.4 ± 0.3	79.5 ± 1.5	220.5 ± 11.9	5.3 ± 0.2
	July 2021	27.1 ± 0.2	26.7 ± 0.2	26.5 ± 0.2	30.2 ± 0.3	84.4 ± 1.0	224.1 ± 12.6	4.7 ± 0.2
	August 2021	27.9 ± 0.2	27.5 ± 0.2	27.1 ± 0.2	30.9 ± 0.2	82.2 ± 0.9	214.1 ± 8.7	4.7 ± 0.2
	September 2021	26.3 ± 0.2	26.2 ± 0.2	25.4 ± 0.1	29.9 ± 0.1	81.7 ± 1.3	240.3 ± 19.3	4.5 ± 0.2

presents the recorded average air temperatures at heights of 60 cm, 2 m, and 10 m above ground, along with soil temperature at a depth of 10 cm, relative humidity at 2 m, solar radiation at 2 m, and wind speed at 10 m.

2.4. Experiment Design and Statistics

Both studies utilized a completely randomized block design, with PGR treatment as the main effect. Each treatment was replicated across three blocks, with each block containing three plants. Statistical analyses were conducted using JMP Pro 16 (SAS Institute, Cary, NC, USA). Statistical calculations were carried out using one-way Analysis of Variance (ANOVA) and Fisher’s Least Significant Difference (LSD). Statistical tests were significant if p < 0.05.

A matrix dataset constructed from the concentrations of the different volatile compound profiles was used for principal component analysis (PCA). MetaboAnalyst (6.0) (Edmonton, AB, Canada) was implemented with Java Server Faces Technology using the Prime Faces library (v13.0) for two-dimensional PCA and visualization.

3. Results and Discussion

3.1. Cone Yield

Little difference in dry cone yield was observed among PGR treatments in Study I (Figure 1). The highest cone yield was observed on plants treated with Ethephon at a lower concentration (50 mg/L) and was 13% higher than the control. In contrast, MaxCel had a greater reduction in cone yield compared to both ProGibb and Ethephon, although these differences were not statistically significant. MaxCel, which contains 6-BA, a synthetic cytokinin, is commonly used in ornamental plants to promote lateral branching. However, it has also been registered for use in fruit thinning applications, where it is designed to reduce overall crop yield while improving individual fruit size and quality. Previous studies have demonstrated that MaxCel can reduce apple crop load by 18–40% while increasing individual fruit size and weight [45,46,47]. It is possible that MaxCel reduced the overall cone yield but increased the individual hop cone size.

Based on the results from Study I, ProGibb and Ethephon were selected for further evaluation in Study II at lower concentrations. In Study II, ProGibb and Ethephon treatment had little effect on dry cone yield (Figure 2). The highest cone yield was observed with a combination of 45 mg/L Ethephon and 3 mg/L ProGibb, resulting in a 125% increase compared to the control. The active ingredient of ProGibb, GA, is commonly used to promote plant height, flower bud formation, and increase fruit or flower yield in the horticulture industry. For instance, applications of 50–75 mg/L GA₃ have been shown to increase flower bud formation by 25–36% and fruit yield by 28–35% in strawberries (Fragaria x ananassa Duch.) [48]. In hops, Bauerle (2022, 2024) reported that applying 5 mg/L of GA more than doubled the number of flowers per plant in the ‘Centennial’ cultivar, but reduced cone mass by 33% [49,50]. The discrepancy between their findings and our study may be attributed to differences in cultivar response. Ethephon, although less commonly used on hops, is typically used to reduce plant height and increase branching in ornamental plants. For example, Kalanchoe marnieriana displayed a 3-fold increase in branching when treated with 1000 mg/L Ethephon compared to a water control [51]. Additionally, Ethephon increased the cutting yield of mother stock, which is directly influenced by lateral branch number, by 22–25% in Impatiens hawkeri and 41–54% in I. walleriana [52]. Interestingly, a combination of GA and Ethephon can be more effective than either PGR used individually. For example, Watanabe et al. demonstrated that a combination of 100 mg/L GA₃ and 50 mg/L Ethephon in rice seedlings resulted in significantly greater plant height, mesocotyl and coleoptile elongation, and larger first and second leaves in deep flooding environments compared to single treatments of GA₃ or Ethephon, as well as untreated controls [53]. This aligns with our findings, where the combination of ProGibb and Ethephon significantly impacted hop cone yield compared to their individual use. This synergy may suggest that the interaction between these PGRs could offer a beneficial strategy for enhancing hop yield.

It is important to note that both studies were conducted on year-1 hop plants. Commercial hop growers typically wait at least two years before harvesting their first crop, allowing the plants to mature and establish a robust root system that supports future growth and yield. However, due to the constraints imposed by the COVID-19 pandemic, only year-1 plants were evaluated in these studies. This limitation may have influenced the overall yield results, as first-year hop plants are known to produce lower yields compared to more established, mature plants. Further research on more established hop plants is needed to fully understand the long-term effects of PGR treatments on hop yield and quality.

3.2. Alpha and Beta Acids

Cascade hops are a widely used American hop variety because of their versatility as a dual-purpose hop. Dual-purpose hops are utilized for their ability to impart bitterness and aromatic qualities depending on what stage they are added to boiling wort [54,55]. Brewers frequently rely on the concentrations of alpha and beta acids in hops to determine dosing quantities to achieve desired bitterness levels in beer. Commercial Cascade hops typically contain alpha acid levels ranging from 4.0% to 9.0%, and beta acids between 5.5% and 9.0%, though these values can vary depending on environmental factors [55].

Table 4. Analysis of alpha and beta acid for ‘Cascade’ hops from Study II.

PGR Treatment	Alpha %	Beta %	Hop Storage Index
Commercial Standard	4.0–9.0 [57]	5.5–9.0 [57]	---
Control (Water)	3.28 (0.21) G	1.59 (0.06) G	0.23 (0.02) A
Ethephon 15 mg/L	3.73 (0.16) F	1.88 (0.09) F	0.02 (0.01) D
Ethephon 30 mg/L	3.24 (0.30) G	2.06 (0.12) E	0.16 (0.01) B
Ethephon 45 mg/L	2.07 (0.09) H	0.87 (0.14) H	0.07 (0.06) D
Ethephon 60 mg/L	2.15 (0.02) H	0.87 (0.06) H	0.14 (0.05) B
ProGibb 1 mg/L	3.84 (0.16) F	1.89 (0.04) F	0.17 (0.01) B
ProGibb 2 mg/L	3.92 (0.12) EF	1.69 (0.02) G	0.14 (0.01) B
ProGibb 3 mg/L	4.08 (0.01) E	2.28 (0.01) D	0.16 (0.01) B
ProGibb 4 mg/L	5.44 (0.04) B	2.32 (0.01) D	0.17 (0.01) B
Ethephon 15 mg/L + ProGibb 1 mg/L	6.09 (0.02) A	2.90 (0.01) A	0.17 (0.01) B
Ethephon 30 mg/L + ProGibb 2 mg/L	5.00 (0.11) C	2.64 (0.08) B	0.15 (0.01) B
Ethephon 45 mg/L + ProGibb 3 mg/L	4.12 (0.17) E	2.48 (0.09) C	0.15 (0.01) B
Ethephon 60 mg/L + ProGibb 4 mg/L	4.51 (0.01) D	1.98 (0.01) EF	0.09 (0.01) CD

N = 3; Means (STD). Values bearing different letters are statistically significant (p < 0.05). --- Not calculated due to reference standards.

compares the alpha and beta acid concentrations for the treatments examined alongside a commercially grown Pacific Northwest Cascade hop as a benchmark. Notably, hop plants treated with water, Ethephon (regardless of concentration), and lower concentrations of ProGibb (1 and 2 mg/L) exhibited alpha acid levels outside the standard commercial range. In contrast, higher concentrations of ProGibb (3 and 4 mg/L), as well as the combination of Ethephon and ProGibb, significantly increased alpha acid content by 24–86% compared to water-treated plants. These treatments brought alpha acid levels within the commercially acceptable range, although still toward the lower to mid-end. This contrasts with the findings of Bauerle (2024), who reported that 5 ppm of GA alone reduced alpha acid levels in ‘Centennial’ hops by 64% compared to untreated controls. Such differences are likely due to cultivar-specific responses [50]. For instance, Zattler and Chrometzka (1968) reported that GA treatments at 5–10 ppm decreased alpha acid content by 15–42% in ‘Hallertauer’ and by 14–33% in ‘Hüller Anfang’, whereas two applications of 10 ppm GA increased alpha acid content by 16% in ‘Saazer’ [56]. These findings highlight the potential of PGR treatments to adjust alpha acid production in a cultivar-dependent manner to meet commercially acceptable standards.

Statistical differences were observed between the different PGR treatments. It should be noted that none of the hops tested contained beta acid levels that fell within the commercial range for this study. These results are similar to other hops that have been grown in Florida [42,58]. The insolubility of β-acids in beer (aqueous) makes them largely irrelevant in the brewing industry [59]. However, the hydrophobicity strength of the β-acids results in a higher bacteriostatic activity when compared to α-acid [60].

3.3. Hop Storage Index

The HSI is an indicator used to measure the degradation of alpha and beta acids during long-term storage and handling of hops [61]. The Hop-6 method [43] measures these acids at two distinct wavelengths, 355 nm and 325 nm, while 275 nm is used to measure degradation products [61]. The calculations for these measurements are shown in Equations (1)–(3) [43]. Both brewers and growers widely employ HSI to assess the freshness and quality of whole cone hops and hop pellets [62,63]. This measurement is crucial because the degradation of alpha acids can lead to significant economic losses for both growers and brewers [26]. However, it is important to recognize that HSI is influenced by various factors such as hop variety, harvest year, timing of the harvest, and kilning conditions [64,65].

HSI values are generally categorized into three ranges: HSI < 0.30 indicates good quality; HSI between 0.30 and 0.40 signifies acceptable quality, with lower values being preferable; and HSI > 0.40 suggests questionable quality [61]. In this study, all tested hops had HSI values below 0.30, confirming they were of good quality (Table 4). While statistical differences were observed between treatments, the fact that all samples fell within the “good quality” range means they would be acceptable for beer production. These findings suggest that although treatments may cause variations in HSI, they do not compromise hop quality if values remain within the acceptable brewing range.

3.4. Volatiles

Hops subjected to various PGR treatments were analyzed for their volatile and semi-volatile aromatic profiles, and the results are summarized in

Table 5. Volatile profile analysis on ‘Cascade’ hops in Study II.

		Approximate Concentration (mg/L)
		Treatments
Compound	LRI Value	1	2	3	4	5	6	7	8	9	10	11	12	13
Acids
Hexadecanoic acid	1930	1.45 (0.41)	0.32 (0.55)	-------	-------	8.15 (1.52)	2.30 (1.09)	4.07 (2.58)	-------	2.43 (4.21)	1.95 (1.39)	-------	6.40 (9.06)	-------
Alcohols
3-Hexenol	840	0.85 (0.10)	0.50 (0.03)	0.88 (0.01)	0.53 (0.08)	-------	-------	-------	-------	-------	0.31 (0.44)	1.97 (0.11)	1.97 (0.11)	-------
Octanol	972	-------	0.15 (0.01)	-------	-------	-------	-------	-------	-------	-------	0.09 (0.12)	2.93 (0.10)	-------	-------
1-Octen-3-ol	976	3.02 (2.64)	0.41 (0.04)	0.97 (0.01)	0.43 (0.02)	1.75 (0.24)	-------	2.99 (0.10)	0.58 (0.08)	3.88 (0.12)	0.63 (0.05)	8.99 (0.18)	2.00 (0.07)	4.53 (0.50)
Benzyl alcohol	1031	0.23 (0.01)	0.13 (0.07)	-------	0.43 (0.10)	-------	-------	-------	-------	-------	-------	-------	0.99 (0.14)	-------
2-Nonanol	1095	-------	-------	0.24 (0.01)	-------	-------	-------	-------	-------	-------	-------	0.35 (0.60)	-------	-------
Subtotal		4.02 BC (2.47)	1.21 CD (0.15)	2.12 BCD (0.01)	1.39 CBD (0.20)	1.75 CBD (0.24)	------- D	2.99 BCD (0.10)	0.58 CD (0.08)	3.88 BC (0.12)	1.02 BCD (0.51)	14.58 A (1.61)	4.96 B (0.18)	4.53 BC (0.50)
Aldehyde
E–2-Pentenal	789	-------	0.28 (0.02)	0.61 (0.01)	-------	-------	-------	-------	-------	-------	-------	-------	-------	-------
2-Hexenal	838	0.99 (0.09)	0.32 (0.02)	0.82 (0.01)	0.12 (0.21)	-------	-------	3.02 (0.43)	-------	2.34 (1.74)	0.47 (0.03)	10.57 (0.60)	0.51 (0.06)	6.68 (0.34)
Benzenecarboxaldehyde	954	0.40 (0.05)	0.23 (0.01)	0.36 (0.01)	0.13 (0.23)	-------	-------	2.10 (0.25)	0.35 (0.06)	2.28 (0.28)	0.34 (0.06)	3.55 (0.03)	2.16 (0.29)	0.73 (1.03)
2,4-Heptadienal	997	-------	-------	-------	-------	-------	-------	-------	-------	-------	-------	4.28 (2.11)	-------	-------
Nonanal	1096	0.63 (0.04)	0.28 (0.23)	-------	-------	-------	-------	4.45 (0.35)	-------	6.69 (0.54)	0.35 (0.06)	18.10 (0.75)	-------	10.02 (1.40)
Subtotal		2.02 DE (0.13)	1.10 DE (0.19)	1.78 D (0.01)	0.25 E (0.44)	-------	-------	9.56 C (0.54)	0.35 DE (0.06)	9.77 C (2.6)	1.16 DE (0.09)	36.50 A (2.12)	2.67 D (0.35)	17.43 B (0.04)
Esters
Methyl hexanoate	921	-------	-------	-------	-------	-------	-------	-------	-------	-------	-------	2.62 (0.14)	-------	-------
Methyl heptanoate	1023	-------	-------	-------	-------	-------	-------	0.68 (0.02)	-------	2.58 (0.18)	-------	8.09 (0.29)	-------	3.91 (0.16)
Methyl octanoate	1118	-------	-------	-------	-------	-------	-------	-------	-------	1.62 (0.34)	-------	3.28 (0.13)	-------	-------
Methyl 4-decenoate	1293	0.99 (0.05)	0.16 (0.01)	0.59 (0.41)	-------	-------	-------	2.54 (0.58)	-------	6.95 (2.69)	-------	18.20 (0.78)	-------	10.78 (0.57)
Methyl geranoate	1304	-------	-------	0.77 (0.01)	-------	-------	-------	1.19 (0.73)	-------	-------	-------	12.94 (0.47)	-------	6.32 (0.62)
Nerol acetate	1363	-------	0.15 (0.02)	2.08 (0.01)	1.13 (0.21)	-------	-------	-------	0.56 (0.12)	12.88 (5.86)	-------	18.60 (0.83)	-------	8.80 (0.95)
Linalyl 3-methylbutanoate	1460	2.15 (0.70)	-------	1.92 (0.01)	-------	-------	-------	-------	-------	5.51 (1.57)	-------	6.61 (0.01)	-------	-------
Geranyl isobutyrate	1501	-------	-------	1.57 (0.01)	0.61 (0.16)	-------	-------	-------	-------	4.74 (0.56)	-------	7.86 (2.66)	-------	-------
5E,7Z-Dodecadienyl acetate	1659	7.41 (2.00)	1.78 (0.33)	3.28 (0.06)	4.62 (0.31)	-------	3.30 (0.75)	2.34 (0.70)	4.05 (0.95)	5.30 (2.19)	1.56 (0.01)	10.56 (0.4)	-------	4.21 ± 5.96
Subtotal		10.55 C (2.75)	2.09 C (0.34)	8.88 C (1.05)	6.16 C (0.32)	-------	3.30 C (0.75)	7.31 C (1.93)	1.24 C (0.29)	34.40 B (6.71)	1.56 C (0.01)	78.64 A (5.41)	-------	34.02 B (8.25)
Ketone
2-Nonanone	1084	0.22 (0.02)	-------	0.22 (0.01)	-------	-------	-------	0.89 (0.03)	-------	1.97 (0.03)	-------	8.89 (0.46)	-------	2.39 (3.38)
2-Decanone	1152	-------	-------	0.26 (0.01)	-------	1.11 (0.01)	-------	-------	-------	-------	-------	5.62 (0.11)	-------	-------
2-Undecanone	1280	1.57 (0.17)	0.35 (0.05)	1.59 (0.01)	-------	-------	0.96 (0.08)	4.26 (1.14)	0.41 (0.02)	7.45 (2.58)	0.41 (0.04)	24.68 (0.86)	0.87 (0.08)	15.91 (1.26)
2-Dodecanone	1343	0.27 (0.07)	-------	0.38 (0.01)	-------	-------	-------	0.56 (0.08)	-------	-------	-------	3.92 (0.48)	-------	-------
β -Ionone	1474	0.32 (0.28)	-------	0.5 (0.01)	-------	-------	-------	-------	-------	-------	-------	2.35 (0.86)	-------	-------
2-Tridecanone	1489	1.52 (0.37)	-------	2.44 (0.01)	0.70 (0.07)	2.82 (0.01)	1.02 (0.21)	3.49 (1.07)	0.82 (0.13)	4.89 (2.70)	-------	19.36 (1.08)	1.49 (0.15)	5.42 (7.67)
Subtotal		3.20 D (0.45)	0.35 D (0.05)	5.37 CD (0.01)	0.70CD (0.07)	3.93 CD (0.01)	1.98 D (0.29)	7.85 CD (3.49)	1.23 D (0.11)	14.00 C (4.35)	0.41 D (0.04)	58.82 A (11.53)	2.36 D (0.07)	23.72 B (9.78)
Monoterpene
β-pinene	977	-------	-------	-------	-------	-------	-------	-------	-------	0.94 (0.40)	-------	1.13 (0.15)	-------	-------
Myrcene	990	-------	-------	-------	-------	-------	-------	11.94 (1.95)	-------	62.44 (3.23)	-------	98.90 (4.73)	-------	55.15 (0.43)
Neral	1233	0.20 (0.01)	-------	0.41 (0.01)	-------	-------	-------	-------	-------	-------	-------	1.62 (0.06)	-------	-------
Citral	1259	0.40 (0.06)	0.12 (0.01)	0.94 (0.01)	0.47 (0.18)	-------	-------	-------	0.41 (0.01)	2.88 (0.87)	0.21 (0.05)	5.61 (0.14)	0.92 (0.04)	-------
Subtotal		0.53 E (0.12)	0.12 E (0.01)	1.22 E (0.31)	0.47 E (0.18)	-------	-------	11.94 D (1.95)	0.41 E (0.01)	65.94 B (3.25)	0.21 E (0.05)	107.26 A (5.04)	0.92 E (0.04)	55.15 C (0.43)
Sequiterpenes
(E)-β caryophyllene	1412	0.31 (0.12)	-------	-------	-------	-------	-------	1.76 (0.21)	-------	9.96 (3.35)	-------	29.90 (1.76)	-------	15.64 (1.66)
γ-Muurolene	1422	-------	-------	-------	-------	-------	-------	-------	-------	-------	-------	2.08 (0.11)	-------	0.74 (1.05)
β-Famesene	1443	-------	-------	-------	-------	-------	-------	0.86 (0.22)	-------	5.99 (2.89)	-------	18.34 (0.94)	-------	8.72 (1.65)
α-Humulene	1452	0.75 (0.23)	-------	0.34 (0.32)	1.21 (1.37)	-------	0.91 (0.44)	4.06 (1.00)	0.35 (0.20)	16.62 (7.30)	0.60 (0.25)	61.10 (3.84)	-------	34.23 (3.30)
Epicubenol	1639	0.74 (0.13)	-------	-------	-------	-------	-------	-------	-------	-------	-------	5.40 (2.26)	-------	6.40 (0.80)
1-Epicadinol	1646	1.49 (0.45)	-------	-------	-------	-------	-------	-------	-------	6.82 (1.96)	-------	14.09 (0.28)	-------	9.22 (1.45)
Subtotal		2.79 C (1.26)	-------	0.34 C (0.32)	1.21 C (1.37)	-------	0.91 C (0.44)	6.10 C (0.44)	0.35 C (0.20)	37.12 B (17.60)	0.60 C (0.25)	126.20 A (3.21)	-------	45.56 B (0.79)
Terepene alcohol
Linalool	1092	2.79 (0.17)	0.91 (0.09)	4.89 (0.31)	2.93 (0.42)	6.54 (0.48)	2.28 (0.42)	2.71 (0.29)	1.64 (0.03)	17.57 (1.47)	1.47 (0.02)	35.06 (1.90)	6.27 (0.67)	13.77 (1.34)
Ipsdienol	1139	-------	-------	0.60 (0.01)	-------	-------	-------	-------	-------	-------	0.24 (0.04)	4.84 (0.28)	-------	-------
4-Terpinenol	1185	-------	-------	-------	-------	-------	-------	-------	-------	-------	-------	3.31 (0.35)	-------	-------
α-Terpineol	1194	1.08 (0.08)	0.64 (0.05)	0.97 (0.01)	0.56 (0.08)	2.18 (0.50)	1.04 (0.41)	2.30 (0.41)	0.66 (0.09)	3.51 (0.67)	0.76 (0.18)	9.00 (1.47)	5.80 (0.81)	4.91 (0.14)
Geraniol	1243	0.73 (0.11)	0.22 (0.06)	2.95 (0.41)	1.58 (0.42)	2.46 (0.59)	-------	-------	0.45 (0.10)	5.39 (4.29)	0.48 (0.05)	12.80 (0.33)	1.80 (0.16)	5.08 (0.02)
α-Selinene	1494	-------	-------	-------	-------	-------	-------	-------	-------	-------	-------	13.09 (1.24)	-------	-------
Subtotal		4.35 EF (0.43)	1.70 F (0.26)	9.21 D (1.25)	5.07 E (0.81)	10.36 CD (1.16)	3.32 EF (0.83)	5.02 E (0.70)	2.75 EF (0.01)	26.47 B (5.39)	2.95 EF (0.11)	77.00 A (3.07)	13.87 C (1.33)	23.77 B (1.45)
Epoxide
Caryophyllene oxide	1588	1.56 (0.42)	0.48 (0.14)	1.75 (0.01)	-------	2.32 (0.28)	1.25 (0.59)	1.90 (0.44)	0.77 (0.15)	4.81 (1.55)	0.75 (0.70)	12.69 (0.75)	1.11 (0.71)	10.31 (1.39)
Humulene epoxide II	1616	2.98 (1.11)	-------	3.30 (0.01)	-------	4.84 (0.13)	-------	3.42 (1.43)	1.44 (0.12)	6.11 (3.76)	-------	22.23 (2.41)	-------	19.91 (2.98)
Caryophyllene oxide II	1663	6.84 (1.82)	1.96 (0.30)	13.41 (1.51)	4.37 (0.35)	12.53 (0.80)	3.79 (0.97)	7.83 (2.73)	2.87 (0.86)	16.19 (10.48)	1.40 (0.24)	41.50 (2.83)	6.30 (1.46)	30.19 (5.23)
Subtotal		10.38 CD (3.46)	2.45 D (0.16)	16.78 BCD (1.41)	4.37 D (0.35)	19.70 BC (0.65)	4.62 D (1.50)	12.02 CD (4.55)	5.08 CD (1.13)	25.51 B (17.12)	2.05 D (0.46)	69.00 A (14.70)	7.41 CD (0.75)	60.41 A (9.60)
Overall Total		39.29 D (4.54)	7.02 D (2.21)	42.91 CD (7.63)	17.01 D (2.21)	32.56 D (13.84)	14.05 D (5.22)	63.94 CD (12.72)	12.00 D (1.57)	216.75 B (47.90)	11.91 D (1.86)	567.66 A (21.02)	39.23 D (9.15)	264.6 BC (9.70)

N = 3; Means (STD). LRI: Linear retention index. Values bearing different letters are statistically significant (p < 0.05). -------: Not Detected. Bold represents the subtotals for that particular organic group and overall totals is the summation of all of the group totals. Treatment 1: Control (water, no-PGR), 2. Ethephon 15 mg/L, 3. Ethephon 30 mg/L, 4. Ethephon 45 mg/L, 5. Ethephon 60 mg/L, 6. ProGibb 1 mg/L, 7. ProGibb 2 mg/L, 8. ProGibb 3 mg/L, 9. ProGibb 4 mg/L, 10. Ethephon 15 mg/L + ProGibb 1 mg/L, 11. Ethephon 30 mg/L + ProGibb 2 mg/L, 12. Ethephon 45 mg/L + ProGibb 3 mg/L. 13. Ethephon 60 mg/L + ProGibb 4 mg/L.

. It should be noted that these values are approximate values; however, the data does show that the utilization of PGRs on the chemical characteristics of hops is impacted by their use. Many of the volatile compounds in hops are derived from plant metabolism or secondary oxidative reactions involving their volatile and non-volatile precursors [66]. Hop volatiles are primarily concentrated in the “hop oil,” a complex mixture composed of hundreds of compounds. The dominant chemical classes in fresh hop oil are monoterpenes (C10 compounds) and sesquiterpenes (C15 compounds), with the most abundant being myrcene, α-humulene, and β-caryophyllene [66]. These three compounds account for roughly 80% of the total volatile profile in hops bred for brewing and were of particular interest in this study [26,67]. Cascade hops, the hops selected for this study, are typically described as having a floral, fruity aroma with citrus notes, often including grapefruit undertones, along with hints of earthy and spicy [54].

As shown in Table 5, the VOCs are tabulated based on chemical groups (alcohols, acids, esters, etc.) with statistics performed between groups, and differences (p < 0.05) were observed. Over 450 volatile compounds have been identified in hop oil, with estimations of over 1000 existing [68]. Despite hops being composed of multiple organic groups, the organic group terpenoids, which can be broken down into terpenes, sesquiterpenes, and the corresponding alcohols, make up the largest percentage of organic compounds in hops [68]. Terpenoid compounds (terpenes, sesquiterpenes, and the corresponding alcohols) made up approximately 54.5% of the total volatile organic profile for Ethephon 30 mg/L + ProGibb 2 mg/L treatment, while only 20% for Ethephon 15 mg/L. Ethephon 30 mg/L + ProGibb 2 mg/L had the highest VOC concentration with 569.67 ± 45.28 mg/L. This treatment led to dramatic increases in both monoterpenes and sesquiterpenes, by approximately 200-fold and 45-fold, respectively, compared to water-treated hops. ProGibb 4 mg/L and Ethephon 60 mg/L + ProGibb 4 mg/L had the second-highest concentrations at 222.83 ± 67.21 and 267.60 ± 30.74 mg/L, respectively. Specifically, monoterpene and sesquiterpene levels increased by 124- and 13-fold under ProGibb 4 mg/L treatment, and by 104- and 16-fold under the Ethephon 60 mg/L + ProGibb 4 mg/L treatment. All the other treatments had significantly lower concentrations compared to ProGibb 4 mg/L and Ethephon 60 mg/L + ProGibb 4 mg/L. Although the effect of PGRs on VOCs in hops remains largely unexplored, studies have shown that GA application can enhance essential oil content and monoterpene synthases expression in aromatic plants such as sage (Salvia officinalis) [69], corn mint (Mentha arvensis) [70], and thyme (Thymus vulgaris) [71].

Previous researchers have identified the essential compounds that contribute to the characteristic “hoppy” flavor in beer. They concluded those compounds were linalool, geraniol, β-damascenone, β-citronellol, esters, and organic acids such as 2- and 3-methylbutanoic acid [22,25,26,27,66,72]. Myrcene is known to dominate the hop oil extract and the aromatic profile of Cascade hops grown in the Pacific Northwest [55,73]. However, the profile of Florida-grown Cascade hops differs significantly. In this study, myrcene made up between 19 and 28% depending on the PGR applied for the total volatile compound profile, much closer to what has been observed in Brazilian-grown Cascade hops [5], as opposed to the 60% typically found in the Pacific Northwest [73]. Other compounds of interest, such as geraniol, were found in all but two treatments above their threshold levels of 14 ng/L [74]. However, while a compound may be found above its threshold level in hop oil, like beer, the aromatic profile of the hop is the sum of parts and not just a single compound [68,75]. Linalool, another compound of interest, was identified in all the treatments and was above its threshold of 3.2 ng/L in water [76]. While several of the compounds of interest, linalool, geraniol, and myrcene, were found in the hop oil extract, it should be noted that the aroma profile of hops is different from the resulting beer [68].

ProGibb (treatment 9) applied at higher concentrations, as well as a combination of Ethephon and ProGibb (treatment 10–13), especially at an application rate of 30 + 2 mg/L (treatment 11), showed to have the greatest potential in achieving the desired volatile compounds that Cascade hops are recognized for. These treatments produced a more balanced profile, potentially enhancing the distinct aroma that Cascade hops are known for in brewing applications. Researchers are continually exploring new regions for hop cultivation, including areas in the United States [77], Brazil [78], and parts of Europe such as Italy and France [79]. Florida’s subtropical to tropical climate presents unique challenges for hop production. However, PGRs offer a promising solution to improve growth and yield without the need for additional field equipment. Further research is needed to understand the underlying mechanisms behind these differences, particularly how environmental conditions, such as those in Florida, interact with PGR treatments to influence volatile production. Understanding these dynamics could lead to optimized cultivation strategies that maintain or enhance the aromatic qualities of hops grown outside traditional regions.

3.5. Principal Component Analysis (PCA)

Based on the alpha and beta acid content presented in Table 4, only treatments of ProGibb at higher concentrations, as well as combinations of Ethephon and ProGibb (treatments 8–13), achieved alpha acid percentages within the commercial standard range. As previously discussed, alpha acid content is one of the primary criteria brewers use when selecting hops, making these six treatments of particular interest.

Figure 3 further details the volatile compound groups correlated with each treatment. Principal Component 1 (PC1) accounts for 42% of the variation in the VOCs among the treatments, while Principal Component 2 (PC2) explains 13.8%. Together, these two components provide insights into the differences in the aromatic profiles associated with each treatment. Ketones—typically contributing sharp or slightly sweet aromas—were primarily associated with the ProGibb 3 mg/L treatment. Aldehydes, which often impart green or grassy aromas, were linked to the Ethephon 15 mg/L + ProGibb 1 mg/L treatment. Terpene alcohols, which can range from sweet and fruity to earthy and pungent depending on their concentration, along with acids that contribute sharp, sour notes, were most strongly associated with the Ethephon 45 mg/L + ProGibb 3 mg/L treatment.

Importantly, fruity, floral, and woody aromas, which are highly desirable characteristics in hop brewing, are typically associated with VOCs such as esters and terpenes. The Ethephon 60 mg/L + ProGibb 4 mg/L treatment was closely linked to esters, which are responsible for fruity aromas. The ProGibb 4 mg/L treatment was primarily correlated with sesquiterpenes, such as α-humulene, known for its earthy and woody scent. Monoterpenes, like myrcene, are often associated with floral and fruity notes and contribute distinctly to the characteristic “hoppy” aroma. The Ethephon 30 mg/L + ProGibb 2 mg/L treatment showed associations with both sesquiterpenes and monoterpenes, indicating a balanced enhancement of these volatile groups. These findings suggest that the Ethephon 30 mg/L + ProGibb 2 mg/L treatment holds the most promise for improving the overall volatile profile of hops compared to untreated controls. Additionally, both ProGibb 4 mg/L and Ethephon 60 mg/L + ProGibb 4 mg/L treatments resulted in substantial increases in VOC concentrations that may benefit hop aroma and flavor. When examined by chemical class, all three treatments significantly enhanced the production of esters (3–7-fold increase), sesquiterpenes (13–45-fold), and monoterpenes (104–202-fold). The latter two are major subgroups of terpenoids—the primary chemical group responsible for hop aroma. These results highlight the potential of specific PGR combinations to markedly enhance the aromatic and flavor qualities of hops for brewing. While it is important to acknowledge that the VOC profile of hops evolves during maturation [55], certain PGR treatments may accelerate this process. Further research is needed to better understand these effects and to optimize PGR application strategies for hop production.

4. Conclusions

Overall, the PGR treatment tested in this study had little effect on the dry cone yield of year-1 hop plants. Combining Ethephon at 45 mg/L and ProGibb at 3 mg/L increased the cone yield by 125% compared to the control. However, PGR treatments significantly changed the chemical composition of the hops. While all treatments had a “good quality” hop storage index, higher concentrations of ProGibb, as well as combinations of Ethephon and ProGibb, produced alpha acid percentages within the commercial standard range for Cascade hops. Specifically, A lower combination of Ethephon and ProGibb (Ethephon 15 mg/L + ProGibb 1 mg/L) had the highest alpha acid percentage, which is best for contributing to bitterness in beer. Additionally, Ethephon 30 mg/L + ProGibb 2 mg/L produced the highest concentration of VOCs at 569.7 mg/L, which is best for contributing to the aroma and flavor of beer. Collectively, our findings suggest that a combination of ProGibb and Ethephon applications can effectively enhance the cone yield and chemical composition of Florida-grown Cascade hops.