Polyphenoles in Saaz hops

---

Dr. ing. Adrian Forster, Hopfenveredelung, St. Johann

Introduction

Many brewers still find polyphenoles from mall or hops undesirable because certain groups of polyphenoles lead to the formation of irreversible haze. To achieve a high physical stability, dosing of polyphenoles is either reduced or partially removed.

Contrary to this, an interesting development has now occurred: both food and plant research now focuses more on searching for naturally occurring anti-oxidants in, for instance, herbs, tea or seeds. The effect of anti-oxidants which often have polyphenolic characters are twofold. On the one hand, they can preserve food items from the negative effects of oxidation and, on the other, they can act as radicals in the human body, i.e. as anti-carcinogens.

The variety Saaz has an excellent reputation as an aroma hop. This reputation is based in part on the noble aroma, the high farnesene contents, the balanced profile in the bitter acids, as well as the high contents of polyphenoles. The statement with respect to polyphenoles is based on non-specific research with colormetric methods (e.g. total polyphenol contents as measured by the EBC-method or antocyanogene according to Harris and Ricketts).

This paper compares the specific polyphenoles of the variety Saaz to other hop varieties. The analyses have been done with a rather involved HPLC method.

Analysis

Until a few years ago, analysis of polyphenoles was possible only using the unspecific colormetric method. In the meantime, much more precise methods have come into existence that use high pressure liquid chromatography (HPLC) coupled with a diode-array-detector (DAD). This allows for the separation of individual components from the polyphenole mixture.

Obr. 1: Chromatogram of polyphenols (characteristics) in Žatec hops at 265 and 310 nm (HPLC method) 1st fraction

The analytical method is as follows: a sample is extracted with an acetone - water mix and then prepared and cleaned with chloroforme. Subsequently, the reduced extract is placed onto a polyamide cartridge. The solid phase extraction is carried out with methanol or alkaline methanol respectively. Both fractions are reduced and membrane filtered prior to injection.

It is recommended to use as eluents for the HPLC a water-formic acid mixture (95/5) as eluent A and a methanol-acetonitril-formic acid mixture (95/5) as eluent B. The process continues from 100 % eluent A via several steps through to 100 % eluent B. The gradient run takes about 70 minutes. It is recommended to use a column of type C18, e. g. Beckman-columns ODS ultrasphere; The detection is done in a UV diode-array-detector (DAD). Approx. 40 individual substances are available for identification and calibration.

Obr. 1: Chromatogram of polyphenols (characteristics) in Žatec hops at 265 and 310 nm (HPLC method) 2nd fraction

Today approx. 100 polyphenole individual components of hops can be separated. The DAD allows a classification of individual groups of substances by permanently recording the spectra. In this way, known peaks can be compared to peaks of the sample. In addition, it is possible to show concurrently data at several wavelengths (e.g. at 265, 280, 310 and 360 nm). In a slightly modified method, one can analyse polyphenoles in both wort and beer.

Figures 1 and 2 show the polyphenole fingerprints of the Saaz variety from two fractions of the polyamide cartridge solid phase extraction at two wavelengths. Table 1 shows the most important substance groups with bandwiths according to the individual contents in hops.

The specific HPLC method results in the sum of the contents of 0.5 - 2.0 volume % which is significantly less than the sum of the total polyphenole values of 3.0 - 6.0 volume %. The reason for this difference is the fact that the HPLC method focuses exclusively on low-molecular components, while the colormetric method includes all medium- and high-molecular components. Another problem with the non-specific analysis is the calibration.

Table 1: Groups of Polyphenols in Hops

Results

The results represent the investigation from hops of crop 1997. The comparison of polyphenoles includes the following varieties:

• Saaz from the growing district Saaz (CZ -SA)
• Tettnang from the growing district Tettnang (TTE), a classic aroma hop with close relationship to the Saaz variety
• Hallertau Mittelfrueh from the growing district Hallertau (HHA), another classic aroma hop
• Hallertau Tradition from the growing district Hallertau (HHT), a new aroma variety from the Huell Hop Research Institute
• Hallertau Magnum from the growing district Hallertau (HHM), a high alpha breeding line also from the Huell Institute

Five lots with good characteristics (visual selection and bitter value) per variety were used and the individual results averaged. Table 2 lists the results of the most important individual components. It is immediately obvious that the individual varieties are quite different. Often a gradient is detected beginning with the high level of the Saaz variety via the other aroma hops down to the Hallertau Magnum which has the lowest values. However, this is not to be regarded as a rule, since there are other qualitative differences. The make up of the polyphenoles, therefore, is not necessarily a function of individual varieties.

Table 2: Polyphenols in 5 Hop varieties; values in mg pro 100g hops (Average of 5 lots - names of varieties see text)

Table 3 lists the totalled groups of substances. Also in this table, it is possible to detect characteristics of varieties. The observations are summarized as below:

• Saaz contains the highest level of most polyphenoles.
• Tettnang is very similar to Saaz. This is very likely the result of its closeness to the Saaz gene pool.
• Hallertau Mittelfrueh is different in several points from Saaz.
• Hallertau Tradition shares common characteristics with Hallertau Mittelfrueh, which is due to its genetic make up of 47 % Hallertau Mittelfrueh. There are also similarities in certain areas to the variety Saaz, which is also included genetically at a level of 15 % in this variety. It is interesting to note that this close genetic relationship is not reflected in the bitter and aroma components. HHT does not contain farnesene.
• The high alpha bitter variety Hallertau Magnum clearly shows the difference between aroma and bitter hops. The make up of the polyphenoles in HHM shows completely different characteristics.

Comparison of Classic Saaz with the Virus Free Saaz

Table 3: Groups of Polyphenols in 5 Hop varieties; values in mg/100 g hops (names of varieties see text)

Using samples from crop 1998, the difference of the polyphenole samples of the classic Saaz and the virus free Saaz are still being investigated. Until now, only data on bitter and aroma components exist. Five samples each were used in this comparison. The calculation of the interval of confidence is shown in table 4. Although the differences are not great, a differentiation nevertheless is possible. This is in contrast to the comparison of the respective aroma and bitter components.

Conclusion

Because polyphenoles are enjoying increasing popularity in the food industry, it would make sense to investigate beer and its raw materials. Using the involved HPLC-DAD analysis, low molecular hop polyphenoles can be separated into approximately 100 individual components.

Significant differences of the make up of low molecular polyphenoles have been found among the varieties Czech Saaz, Tettnang Tettnang, Hallertau Hallertau Mittelfrueh.

The relationship of Tettnang to the Saaz gene pool can be found. With respect the Hallertau Tradition it is possible to detect a relationship via the polyphenoles to not only Hallertau Hallertau (47% of the genetic make up) but also Saaz (15% of the genetic make up). A high alpha variety, such as the Hallertau Magnum, shows a level of polyphenoles much below that of an aroma variety. Significant qualitative differences have also been found.

Table 4: Mean value (MV) and Interval of confidence (IC) of some Polyphenoles of classical and virus free Saaz hop; (values in mg/100 g hops as is)

The classic and virus free Saaz show several differences in their respective polyphenole sample. The specific HPLC analysis that aims at the separation of low molecular polyphenoles is an efficient method in describing varieties. In addition, qualitative conclusions can be drawn.

In general terms, the classical Saaz aroma hop is a variety with one of the highest amounts of low molecular polyphenoles.