Interesting Behavior of Geranic Acid during the Beer Brewing Process: Why Could Geranic Acid Remain at a Higher Level Only in the Beer Using Sorachi Ace Hops?

Hops are among the most important ingredients in beer that contribute to beer flavor. Consequently, novel types of hops have been bred and widely used worldwide. For example, the Sorachi Ace hop imparts characteristic varietal aromas, including woody, pine-like, citrus, dill-like, and lemongrass-like aromas, to the finished beer. In our previous study, the unique volatile compound geranic acid was significantly detected only in the test beer brewed with the Sorachi Ace hop; moreover, the coexistence of geranic acid and other hop-derived flavor compounds could result in the characteristic aroma of the Sorachi Ace beers. In this study, selected hop-derived flavor compounds, including geranic acid, were compared among 17 hop varieties. The geranic acid content in the Sorachi Ace hop was the highest among the studied hops. We also investigated the behavior of geranic acid and related flavor compounds throughout the fermentation process. The content of geranic acid was higher than those of the other compounds during fermentation. Next, we compared the concentrations of these compounds in kettle-, late-, and dry-hopped beers using Sorachi Ace hop. The results revealed that geranic acid remained at higher concentrations from the worts to finished beers despite the decrease in the content of other hop-derived flavor compounds as a result of evaporation and/or other factors during brewing. Further, geranic acid could remain at high levels in the test-brewed beers with Sorachi Ace hops because of its behavior as an acid throughout the brewing process, including during wort boiling and fermentation.

Development of a flavor hop (Humulus lupulus L.) cultivar, 'Furano Magical', with cones rich in 4-methyl-4-sulfunylpentan-2-one.

BACKGROUND: Flavor hops (Humulus lupulus L.) are recognized as key raw materials that impart unique flavors to beer, especially in the emerging craft-beer industry. In this study the sensory evaluation of hop cone aromas using hop water extract by boiling (HWEB) was applied to develop new flavor hop cultivars in a screening from tens of varieties and breeding lines. RESULTS: The sensory scores of HWEB showed a significant correlation with polyfunctional thiol content in hop cones, including substances such as 4-methyl-4-sulfunylpentan-2-one (4MSP) and 3-sulfunyl-4-methylpentan-1-ol (3S4MP), which are known to be difficult to analyze mechanically. As a result of the sensory evaluation of HWEB, a breeding line, 'K906901060' was found to have a strong fruity note. Subsequent chemical analyses revealed that this cultivar had 80-136 µg kg-1 of 4MSP in its cones, which is similar to the concentration present in current leading flavor hop cultivars, such as Citra (37-114 µg kg-1 ) and Simcoe (2-112 µg kg-1 ). Beer late-hopped with K906901060 provoked a greater tropical flavor impression than beer hopped using Nelson Sauvin hops. CONCLUSION: The sensory evaluation of HWEB was indicated to be useful to search for hops containing polyfunctional thiols. The plant was registered, as 'Furano K906901060 Go' in Japan, in the EU, and in the USA, and the cultivar was given the commercial name 'Furano Magical'. This cultivar was derived from a cross performed in 1989 and had been kept as a mid-mother plant from 1997, until being selected in 2014, as described here. © 2022 Society of Chemical Industry.

Effect of "Late Harvest" of Hops (Humulus lupulus L.) on the Contents of Volatile Thiols in Furano Beauty, Furano Magical, and Cascade Varieties.

In recent years, many hop varieties with unique aromas, so-called "flavor hops", have been bred and grown. Here, we investigated the effect of late-harvested hops using three flavor hop varieties; Furano Beauty, Furano Magical, and Cascade. The sample hops were harvested at different days after flowering (DAF): DAF 45 (normal harvest), DAF 65, DAF 75, and DAF 85. We measured the volatile thiols in sample cones. The results indicated that 4-methyl-4-sulfanylpentane-2-one contents showed almost no change or a slightly decrease with a delay in harvest, whereas 3-sulfanyl-4-methylpentan-1-ol (3S4MP) content in late-harvested samples increased several fold in comparison with normal-harvested samples. Additionally, 3S4MP contents in the beers brewed with DAF 65 samples were several times higher than those using DAF 45 ones. From these results, we propose a new method to control 3S4MP content in hop cones by changing its harvest date.

Identification and Characterization of Geranic Acid as a Unique Flavor Compound of Hops ( Humulus lupulus L.) Variety Sorachi Ace.

Hops are natural ingredients used to impart bitterness and flavor to beer. Recently, new varieties of hops have attracted global research attention. The Sorachi Ace variety, in particular, interests many craft brewers. This hop imparts characteristic varietal aromas, including woody, pine-like, citrus, dill-like, and lemongrass-like, to finished beers. Here, we investigated specific flavor compounds derived from Sorachi Ace using selectable one-dimensional or two-dimensional gas chromatography-olfactometry/mass spectrometry and head space-solid phase microextraction-gas chromatography-mass spectrometry. The results showed that a unique volatile compound, geranic acid, was present at a significant level only in the test beer brewed with the Sorachi Ace hop. Furthermore, sensory evaluation techniques revealed that geranic acid has very unique characteristics. This compound is not odor-active but functions as an enhancer for hop-derived terpenoids at subthreshold levels.

Quantitation Method for Polyfunctional Thiols in Hops (Humulus lupulus L.) and Beer Using Specific Extraction of Thiols and Gas Chromatography-Tandem Mass Spectrometry.

A method for the quantitation of six polyfunctional thiols, 4-methyl-4-sulfanylpentan-2-one (4MSP), 3-sulfanyl-4-methylpentan-1-ol (3S4MP), 3-sulfanyl-4-methylpentyl acetate (3S4MPA), 3-sulfanyl-3-methylbutan-1-ol (3S3MB), 3-sulfanylhexan-1-ol (3SH), and 3-sulfanylhexyl acetate (3SHA), in hops and beer without organic mercury compounds was developed. The method employed specific extraction of thiols using a silver ion solid phase extraction (SPE) cartridge and gas chromatography-tandem mass spectrometry (GC-MS/MS). For all thiols analyzed, good linearity was achieved by adding thioglycerol as an analyte protectant. Recoveries for both hops (74-100%) and beer (79-113%) were acceptable, and the repeatability for both was also good (relative standard deviations of 2.8-8.4%). The limits of detection for the six polyfunctional thiols were below their odor thresholds in beer. The method was applied to quantitation of hops and beer flavored with thiol-containing hop varieties. Due to their detected levels and level variations in different beers, 4MSP and 3S4MP are thought to be important polyfunctional thiols for the characteristic flavor of hop varieties.

Biotransformation of hop-derived monoterpene alcohols by lager yeast and their contribution to the flavor of hopped beer.

It is well-known that various beers contain many flavor compounds derived from barley malts, hops, yeast fermentation, and other raw materials. Among these flavor compounds, terpenoids are mainly derived from hops. Linalool, one of the monoterpene alcohols, has been found in various beers and been regarded as an important factor for a hop-derived beer flavor. We focus on contributions of other monoterpene alcohols (geraniol, beta-citronellol, nerol, and alpha-terpineol) to hopped beer flavor. Several researchers have reported that monoterpene alcohols are biotransformed by yeast and that geraniol is mainly transformed to beta-citronellol during the first 2-4 days in model fermentation. In this study, we investigated the biotransformation of monoterpene alcohols during fermentation of hopped beer by using various hop cultivars. As a result, geraniol drastically decreased during the first 3 days. beta-Citronellol was almost absent in wort and gently increased during the total fermentation period. The concentrations of geraniol and beta-citronellol in finished beer increased, depending on the initial concentration of geraniol in the wort. The continuous increase of beta-citronellol did not correspond to the fast decrease of geraniol. This increase of beta-citronellol might be partly explained by an occurrence of glycosidically bound flavor precursor and a glucoside hydrolase activity secreted from lager yeast. In addition, we examined flavor characteristics of monoterpene alcohols and found that there was an additive effect among linalool, geraniol, and beta-citronellol and that only 5 microg/L of geraniol and beta-citronellol were enough for this effect. Therefore, it is suggested that not only linalool but also geraniol and beta-citronellol might contribute to hopped beer flavor at lower levels, at which OAVs of these compounds become below 1.0.

Identification and characteristics of new volatile thiols derived from the hop (Humulus luplus L.) cultivar Nelson Sauvin (dagger).

Nelson Sauvin (NS) is a unique hop cultivar that was bred and grown in New Zealand. This hop gives a specific flavor (exotic fruit-like, white wine-like) to finished beers. However, the key compounds of this flavor have not yet been identified. We have attempted to identify the specific flavor compounds derived from NS. We focused on certain volatile thiols that are well-known to contribute to wine flavors, especially Sauvignon Blanc. The product made from NS (NS product) lost its specific flavor by contact with copper. Copper is well-known as an absorber of thiols in the field of wine flavor investigations. Therefore, it might point to the existence of thiols. We analyzed the NS product by GC-FPD, GC-olfactometry and GC-MS, and identified two new volatile thiols, 3-sulfanyl-4-methylpentan-1-ol (3S4MP), and 3-sulfanyl-4-methylpentyl acetate (3S4MPA). These compounds have a grapefruit-like and/or rhubarb-like odor, similar to that of Sauvignon Blanc. We quantified these compounds in the NS products and determined their thresholds. As a result, 3S4MP contained about 2-fold of its threshold in beers, and 3S4MPA was included below its threshold. However, it was confirmed that 3S4MP enhanced the flavors of 3S4MPA by synergy. Therefore, we concluded that both of the new volatile thiols would contribute to the specific odor of beers produced with NS.

Novel prediction method of beer foam stability using protein Z, barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and yeast thioredoxin.

Foam stability is an important quality trait of beer. Our previous results of two-dimensional gel electrophoresis (2DE) analyses of beer proteins implied a relationship between barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and beer foam stability as judged by the NIBEM-T analyzer. To develop a novel prediction method of beer foam stability under different conditions of barley cultivar and malt modification, multiple linear regression analysis was applied. The spot intensities of major beer proteins on 2DE gel were quantified and used as explanatory variables. The foam stabilities of 25 beer samples each brewed from malt with different malt modification in one of the three cultivars (cultivars A, B, and C) were explained by the spot intensities of BDAI-1 at the 5% significance level ( r = 0.421). Furthermore, two other major protein spots (b0 and b5) were observed on the 2DE gels of Japanese commercial beer samples with different foam stability. Then, multiple regression for foam stability was calculated using these three spot intensities as explanatory variables. As a result, 72.1% of the beer foam stability in 25 beer samples was explained by a novel multiple regression equation calculated using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. To verify the validity of the multiple regression equation and the explanatory variables, the beer foam stability in practical beer samples was analyzed. As a result, 81.5% of the beer foam stability in 10 Japanese commercial beer samples was also explained by using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. Mass spectrometry analyses followed by database searches revealed that protein spots b0 and b5 were identified as protein Z originated from barley and thioredoxin originated from yeast, respectively. These results confirm that BDAI-1 and protein Z are foam-positive factors and identify yeast thioredoxin as a possible novel foam-negative factor.

The influence of barley malt protein modification on beer foam stability and their relationship to the barley dimeric alpha-amylase inhibitor-I (BDAI-I) as a possible foam-promoting protein.

The foam stability of beer is one of the important key factors in evaluating the quality of beer. The purpose of this study was to investigate the relationship between the level of malt modification (degradation of protein, starch, and so on) and the beer foam stability. This was achieved by examining foam-promoting proteins using two-dimensional gel electrophoresis (2DE). We found that the foam stability of beer samples brewed from the barley malts of cultivars B and C decreased as the level of malt modification increased; however, the foam stability of cultivar A did not change. To identify the property providing the increased foam stability of cultivar A, we analyzed beer proteins using 2DE. We analyzed three fractions that could contain beer foam-promoting proteins, namely, beer whole proteins, salt-precipitated proteins, and the proteins concentrated from beer foam. As a result, we found that in cultivar A, some protein spots did not change in any of these three protein fractions even when the level of malt modification increased, although the corresponding protein spots in cultivars B and C decreased. We analyzed these protein spots by peptide mass finger printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. As a result, all of these spots were identified as barley dimeric alpha-amylase inhibitor-I (BDAI-I). These results suggest that BDAI-I is an important contributor to beer foam stability.