Hop Isomerization - Revision history

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2016-08-03T12:09:12Z

Author

Bes at 12:08, 3 August 2016

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Bes at 12:06, 3 August 2016

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Chrisk: /* References */

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References

Chrisk: /* Results */

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Results

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Results

Chrisk: /* Materials and Methods */

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Materials and Methods

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Materials and Methods

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Introduction

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Introduction

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 =Author=
-[[Chris_Knutson|Christopher J. Knutson]] Monmouth College, May 2016.
+[[Chris_Knutson|Christopher J. Knutson]] Monmouth College, Department of Chemistry, May 2016.
 =Abstract=

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 =Author=
-Christopher J. Knutson
+[[Chris_Knutson|Christopher J. Knutson]] Monmouth College, May 2016.
 =Abstract=

← Older revision		Revision as of 12:06, 3 August 2016
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		+	=Author=
		+	Christopher J. Knutson
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	=Abstract=		=Abstract=
	Hops are added to the brew kettle at different times in order to add specific character to the beer. The addition of hops early in the boiling process (early addition) primarily add bitterness; while the addition of hops late in the boiling process (late addition) primarily flavor and aroma. Hop bitterness comes from the isomerization of α-acids with effective utilization of not more than 30%. The reason for low utilization is most likely a combination of factors related to both the volatility of the α-acids as well as degradation of the isomerized α-acids. The contribution of late addition hops to the final beer is a result of essential oils and is not fully understood. Recipe formulations which have a strong hop flavor component add hops throughout the brewing process as well as after primary fermentation (dry hopping). This research developed a method of pre-isomerizing hop α-acids in order to increase utilization efficiency as well as a method of high performance liquid chromatography (HPLC) analysis of both the α-acids and iso-α-acids during a single injection. The data presented will show that hop utilization can be improved substantially through simply heat treating the hops before addition to the brewing process and that the isomerization reaction can be characterized using a universal HPLC method for both the starting and end products of the reaction.		Hops are added to the brew kettle at different times in order to add specific character to the beer. The addition of hops early in the boiling process (early addition) primarily add bitterness; while the addition of hops late in the boiling process (late addition) primarily flavor and aroma. Hop bitterness comes from the isomerization of α-acids with effective utilization of not more than 30%. The reason for low utilization is most likely a combination of factors related to both the volatility of the α-acids as well as degradation of the isomerized α-acids. The contribution of late addition hops to the final beer is a result of essential oils and is not fully understood. Recipe formulations which have a strong hop flavor component add hops throughout the brewing process as well as after primary fermentation (dry hopping). This research developed a method of pre-isomerizing hop α-acids in order to increase utilization efficiency as well as a method of high performance liquid chromatography (HPLC) analysis of both the α-acids and iso-α-acids during a single injection. The data presented will show that hop utilization can be improved substantially through simply heat treating the hops before addition to the brewing process and that the isomerization reaction can be characterized using a universal HPLC method for both the starting and end products of the reaction.

← Older revision		Revision as of 20:46, 19 April 2016
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	=References=		=References=

−	~~# ASBC Methods of Analysis. Hops Method 14. Approved 1990. American Society of Brewing Chemists, St. Paul, MN, U.S.A. doi: 10.1094~~/~~ASBCMethod-Hops14~~	+	<references />
−	~~# Danenhower, T. M.; Force, L. J.; Peterson, K. J.; Betts, T. A.; Baker, G. A. HPLC Analysis of α- and β-Acids in Hops. J. Chem. Ed. 2008, 85(7), 954-956.~~
−	~~# De Keukelleire, D. Fundamentals of Beer and Hop Chemistry. Quimica Nova, 2000, 23(1), 108-112.~~
−	~~# Kappler, S.; Kral, M.; Geissinger, C.; Becker, T.; Krottenthaler, M. Degradation of Iso-a-Acids During Wort Boiling. J. Inst. Brew. 2010, 116(4), 332-338.~~
−	~~# Malowicki, M. G. and Shellhammer, T. H. Isomerization and Degradation Kinetics of Hop (Humulus lupulus) Acids in a Model Wort-Boiling System. J. Ag. Food Chem. 2005, 53, 4434-4439.~~
−	~~# Urban, J.; Dahlberg, C. J.; Carroll, B. J.; Kaminsky, W. Absolute Configuration of Beer’s Bitter Compounds. Angew. Chem. Int. Ed. 2013, 52, 1553-1555.~~
−	~~# Biendl, M. Pre-isomerised Hop Products - Potential and Practical Use. Brauwelt International, 2002, 20–25.~~

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 Significant time was invested in HPLC methodology development in order to optimize peak resolution on the chromatograph. The primary challenge was developing a method which would allow the characterization of both the α-acids and iso-α-acids. (Figure 2) A gradient method was ultimately developed which would allow a single trial to be completed within 35 minutes on a waters HPLC System using a Supelco Analytical Supelcosil LC-18 25cm 4.6mm, 5mm HPLC column. 10 μl injections were introduced to the column at 28 ⁰C with a gradient mobile phase: 65% MeOH 0-12 min, then up to 85% MeOH by 18 min, hold at 85% until 28 min, then back to 65% MeOH at 33 min. UV detection was focused on peak resolution at 270nm.<ref name="ASBC" /><ref name="Danenhower" />
-[[File:Results.jpg|thumb|500px|frame|center|'''Figure 2:''' Reverse-phase high performance liquid chromatography (HPLC) with UV detection at 270 nm chromatographs depicting ASBC standards in comparison to a heat treated sample of Chinook hops. A Waters HPLC System was employed using a Supelco Analytical Supelcosil LC-18 25cm 4.6mm, 5mm HPLC column. 10 μl injections were introduced to the column at 28 ⁰C with a gradient mobile phase: 65% MeOH 0-12 min, then up to 85% MeOH by 18 min, hold at 85% until 28 min, then back to 65% MeOH at 33 min. 1,2]]
+[[File:Results.jpg|thumb|500px|frame|center|'''Figure 2:''' Reverse-phase high performance liquid chromatography (HPLC) with UV detection at 270 nm chromatographs depicting ASBC standards in comparison to a heat treated sample of Chinook hops. A Waters HPLC System was employed using a Supelco Analytical Supelcosil LC-18 25cm 4.6mm, 5mm HPLC column. 10 μl injections were introduced to the column at 28 ⁰C with a gradient mobile phase: 65% MeOH 0-12 min, then up to 85% MeOH by 18 min, hold at 85% until 28 min, then back to 65% MeOH at 33 min. <ref name="ASBC" /><ref name="Danenhower" /> ]]
 Once the HPLC method was confirmed through acceptable peak resolution of hop standards, analysis was conducted on Chinook samples which had been previously heat treated and extracted. The HPLC chromatographs depict α and β acid content of Chinook Hop sample extractions. (Figure 3) The α and β acid peaks elute around the 20 minute mark. The first two peaks, of four, around the 20 minute mark are attributed to α-acids. The final two peaks are β-acids. This series of chromatographs show a reduction of α-acid upon heat treatment that is dependent on the length of time a particular sample is exposed to heat. In conjunction with the reduction of α-acids, these chromatographs also show an increase in sub 10 minute peaks attributable to the formation of iso-α-acids.

← Older revision		Revision as of 20:44, 19 April 2016
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−	Significant time was invested in HPLC methodology development in order to optimize peak resolution on the chromatograph. The primary challenge was developing a method which would allow the characterization of both the α-acids and iso-α-acids. (Figure 2) A gradient method was ultimately developed which would allow a single trial to be completed within 35 minutes on a waters HPLC System using a Supelco Analytical Supelcosil LC-18 25cm 4.6mm, 5mm HPLC column. 10 μl injections were introduced to the column at 28 ⁰C with a gradient mobile phase: 65% MeOH 0-12 min, then up to 85% MeOH by 18 min, hold at 85% until 28 min, then back to 65% MeOH at 33 min. UV detection was focused on peak resolution at 270nm.~~1, 2~~	+	Significant time was invested in HPLC methodology development in order to optimize peak resolution on the chromatograph. The primary challenge was developing a method which would allow the characterization of both the α-acids and iso-α-acids. (Figure 2) A gradient method was ultimately developed which would allow a single trial to be completed within 35 minutes on a waters HPLC System using a Supelco Analytical Supelcosil LC-18 25cm 4.6mm, 5mm HPLC column. 10 μl injections were introduced to the column at 28 ⁰C with a gradient mobile phase: 65% MeOH 0-12 min, then up to 85% MeOH by 18 min, hold at 85% until 28 min, then back to 65% MeOH at 33 min. UV detection was focused on peak resolution at 270nm.<ref name="ASBC" /><ref name="Danenhower" />

	[[File:Results.jpg\|thumb\|500px\|frame\|center\|'''Figure 2:''' Reverse-phase high performance liquid chromatography (HPLC) with UV detection at 270 nm chromatographs depicting ASBC standards in comparison to a heat treated sample of Chinook hops. A Waters HPLC System was employed using a Supelco Analytical Supelcosil LC-18 25cm 4.6mm, 5mm HPLC column. 10 μl injections were introduced to the column at 28 ⁰C with a gradient mobile phase: 65% MeOH 0-12 min, then up to 85% MeOH by 18 min, hold at 85% until 28 min, then back to 65% MeOH at 33 min. 1,2]]		[[File:Results.jpg\|thumb\|500px\|frame\|center\|'''Figure 2:''' Reverse-phase high performance liquid chromatography (HPLC) with UV detection at 270 nm chromatographs depicting ASBC standards in comparison to a heat treated sample of Chinook hops. A Waters HPLC System was employed using a Supelco Analytical Supelcosil LC-18 25cm 4.6mm, 5mm HPLC column. 10 μl injections were introduced to the column at 28 ⁰C with a gradient mobile phase: 65% MeOH 0-12 min, then up to 85% MeOH by 18 min, hold at 85% until 28 min, then back to 65% MeOH at 33 min. 1,2]]

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 *1% Concentrated HCl
-The extractions were mechanically stirred for 1 hour and then filtered through a 0.45 μm filter and introduced to a Supelco Analytical Supelcosil LC-18 25cm 4.6mm, 5mm HPLC column in 10 μl injections. High performance liquid chromatography was conducted at 28 ⁰C with a gradient mobile phase: 65% MeOH 0-12 min, then up to 85% MeOH by 18 min, hold at 85% until 28 min, then back to 65% MeOH at 33 min. <ref name="ASBC" /><ref name="Danenhower">
+The extractions were mechanically stirred for 1 hour and then filtered through a 0.45 μm filter and introduced to a Supelco Analytical Supelcosil LC-18 25cm 4.6mm, 5mm HPLC column in 10 μl injections. High performance liquid chromatography was conducted at 28 ⁰C with a gradient mobile phase: 65% MeOH 0-12 min, then up to 85% MeOH by 18 min, hold at 85% until 28 min, then back to 65% MeOH at 33 min. <ref name="ASBC" /><ref name="Danenhower" />
 =Results=

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 Hops can be a major financial investment in a beer. The wholesale price of grains (~$0.50/lb.; $1/kg) is considerably less than the price of hops (~$10/lb.; $20/kg). In the above example hops are ~1% of the recipe but cost ~2000% (x20) more than grain. As above, for every 1 US gallon (~4 L) of beer, $1 of grains and $0.25 of hops are needed. As with most business related activities, increasing the process efficiency is one means of lowering the production costs.
-Under standard brewing conditions, an estimate of 30% of the added a-acids are converted into iso-α-acids. The low utilization efficiency is a result of many factors including, the volatility of α-acids, the degradation of iso-α-acids, and the physical dynamics of the brewing process.<ref name="Keukelleire">De Keukelleire, D. Fundamentals of Beer and Hop Chemistry. Quimica Nova, 2000, 23(1), 108-112.</ref><ref name="Kappler">Kappler, S.; Kral, M.; Geissinger, C.; Becker, T.; Krottenthaler, M. Degradation of Iso-a-Acids During Wort Boiling. J. Inst. Brew. 2010, 116(4), 332-338.</ref><ref name="Malowicki" />   The current strategy of increasing hop utilization in large scale industrial brewing is to mix hop pellets with a catalyst, such as magnesium oxide, and allow the hops to rest for a 8-12 days at 50C.<ref name="Biendl">Biendl, M. Pre-isomerised Hop Products - Potential and Practical Use. Brauwelt International, 2002, 20–25.</ref> This research sought to produce a pre-isomerization method for a small scale application which would not require the use of additional catalysts. We have undertaken a set of experiments to determine if we can pre-isomerize α-acids in the hop cone prior to the brewing process. Our approach involves the heating of packaged hop material at 100 ⁰C for 1,2,3,4 hours. Analysis of the α-acids and iso-α-acids is done by high performance liquid chromatography (HPLC).<ref name="ASBC">ASBC Methods of Analysis. Hops Method 14. Approved 1990. American Society of Brewing Chemists, St. Paul, MN, U.S.A. doi: 10.1094/ASBCMethod-Hops14</ref><ref name="Danenhower">Danenhower, T. M.; Force, L. J.; Peterson, K. J.; Betts, T. A.; Baker, G. A. HPLC Analysis of α- and β-Acids in Hops. J. Chem. Ed. 2008, 85(7), 954-956.</ref>
+Under standard brewing conditions, an estimate of 30% of the added a-acids are converted into iso-α-acids. The low utilization efficiency is a result of many factors including, the volatility of α-acids, the degradation of iso-α-acids, and the physical dynamics of the brewing process.<ref name="Keukelleire">De Keukelleire, D. Fundamentals of Beer and Hop Chemistry. Quimica Nova, 2000, 23(1), 108-112.</ref><ref name="Kappler">Kappler, S.; Kral, M.; Geissinger, C.; Becker, T.; Krottenthaler, M. Degradation of Iso-a-Acids During Wort Boiling. J. Inst. Brew. 2010, 116(4), 332-338.</ref><ref name="Malowicki" />   The current strategy of increasing hop utilization in large scale industrial brewing is to mix hop pellets with a catalyst, such as magnesium oxide, and allow the hops to rest for a 8-12 days at 50⁰C.<ref name="Biendl">Biendl, M. Pre-isomerised Hop Products - Potential and Practical Use. Brauwelt International, 2002, 20–25.</ref> This research sought to produce a pre-isomerization method for a small scale application which would not require the use of additional catalysts. We have undertaken a set of experiments to determine if we can pre-isomerize α-acids in the hop cone prior to the brewing process. Our approach involves the heating of packaged hop material at 100 ⁰C for 1,2,3,4 hours. Analysis of the α-acids and iso-α-acids is done by high performance liquid chromatography (HPLC).<ref name="ASBC">ASBC Methods of Analysis. Hops Method 14. Approved 1990. American Society of Brewing Chemists, St. Paul, MN, U.S.A. doi: 10.1094/ASBCMethod-Hops14</ref><ref name="Danenhower">Danenhower, T. M.; Force, L. J.; Peterson, K. J.; Betts, T. A.; Baker, G. A. HPLC Analysis of α- and β-Acids in Hops. J. Chem. Ed. 2008, 85(7), 954-956.</ref>
 =Materials and Methods=

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 Hops are added during the brewing process to contribute to three main characteristics to the beer: bitterness, flavor, and aroma. To achieve these characteristics, different hop varieties are selected and the hops are added to the boil kettle at different times. Hops added “early” in the 60 minute boil add bitterness; hops added “late” contribute to the flavor and aroma. The chemical composition of hops that add flavor and aroma are associated with essential oils that reside in the Lupulin glands of the hop flower (cone).Hops added early also contain these essential oils, but these oils are volatilized in the boil kettle.<ref name="Malowicki">Malowicki, M. G. and Shellhammer, T. H. Isomerization and Degradation Kinetics of Hop (Humulus lupulus) Acids in a Model Wort-Boiling System. J. Ag. Food Chem. 2005, 53, 4434-4439.</ref>
-It is understood that the bitterness associated with hops added early in the wort boiling process is a product of the isomerization of α-acids within the hop. Non-isomerized α-acids typically have a low solubility; however, once isomerized, α-acids take on a much more soluble iso-α-acid form.<ref name="Urban">Urban, J.; Dahlberg, C. J.; Carroll, B. J.; Kaminsky, W. Absolute Configuration of Beer’s Bitter Compounds. Angew. Chem. Int. Ed. 2013, 52, 1553-1555.</ref>(Figure 1).
+It is understood that the bitterness associated with hops added early in the wort boiling process is a product of the isomerization of α-acids within the hop. Non-isomerized α-acids typically have a low solubility; however, once isomerized, α-acids take on a much more soluble iso-α-acid form.<ref name="Urban">Urban, J.; Dahlberg, C. J.; Carroll, B. J.; Kaminsky, W. Absolute Configuration of Beer’s Bitter Compounds. Angew. Chem. Int. Ed. 2013, 52, 1553-1555.</ref>(Figure 1)
 Hops can be a major financial investment in a beer. The wholesale price of grains (~$0.50/lb.; $1/kg) is considerably less than the price of hops (~$10/lb.; $20/kg). In the above example hops are ~1% of the recipe but cost ~2000% (x20) more than grain. As above, for every 1 US gallon (~4 L) of beer, $1 of grains and $0.25 of hops are needed. As with most business related activities, increasing the process efficiency is one means of lowering the production costs.
-Under standard brewing conditions, an estimate of 30% of the added a-acids are converted into iso-α-acids. The low utilization efficiency is a result of many factors including, the volatility of α-acids, the degradation of iso-α-acids, and the physical dynamics of the brewing process.<ref name="Keukelleire">De Keukelleire, D. Fundamentals of Beer and Hop Chemistry. Quimica Nova, 2000, 23(1), 108-112.</ref>,<ref name="Kappler">Kappler, S.; Kral, M.; Geissinger, C.; Becker, T.; Krottenthaler, M. Degradation of Iso-a-Acids During Wort Boiling. J. Inst. Brew. 2010, 116(4), 332-338.</ref>,<ref name="Malowicki" />   The current strategy of increasing hop utilization in large scale industrial brewing is to mix hop pellets with a catalyst, such as magnesium oxide, and allow the hops to rest for a 8-12 days at 50C.7 This research sought to produce a pre-isomerization method for a small scale application which would not require the use of additional catalysts. We have undertaken a set of experiments to determine if we can pre-isomerize α-acids in the hop cone prior to the brewing process. Our approach involves the heating of packaged hop material at 100 ⁰C for 1,2,3,4 hours. Analysis of the α-acids and iso-α-acids is done by high performance liquid chromatography (HPLC).<ref name="ASBC">ASBC Methods of Analysis. Hops Method 14. Approved 1990. American Society of Brewing Chemists, St. Paul, MN, U.S.A. doi: 10.1094/ASBCMethod-Hops14</ref>,<ref name="Danenhower">Danenhower, T. M.; Force, L. J.; Peterson, K. J.; Betts, T. A.; Baker, G. A. HPLC Analysis of α- and β-Acids in Hops. J. Chem. Ed. 2008, 85(7), 954-956.</ref>
+Under standard brewing conditions, an estimate of 30% of the added a-acids are converted into iso-α-acids. The low utilization efficiency is a result of many factors including, the volatility of α-acids, the degradation of iso-α-acids, and the physical dynamics of the brewing process.<ref name="Keukelleire">De Keukelleire, D. Fundamentals of Beer and Hop Chemistry. Quimica Nova, 2000, 23(1), 108-112.</ref><ref name="Kappler">Kappler, S.; Kral, M.; Geissinger, C.; Becker, T.; Krottenthaler, M. Degradation of Iso-a-Acids During Wort Boiling. J. Inst. Brew. 2010, 116(4), 332-338.</ref><ref name="Malowicki" />   The current strategy of increasing hop utilization in large scale industrial brewing is to mix hop pellets with a catalyst, such as magnesium oxide, and allow the hops to rest for a 8-12 days at 50C.<ref name="Biendl">Biendl, M. Pre-isomerised Hop Products - Potential and Practical Use. Brauwelt International, 2002, 20–25.</ref> This research sought to produce a pre-isomerization method for a small scale application which would not require the use of additional catalysts. We have undertaken a set of experiments to determine if we can pre-isomerize α-acids in the hop cone prior to the brewing process. Our approach involves the heating of packaged hop material at 100 ⁰C for 1,2,3,4 hours. Analysis of the α-acids and iso-α-acids is done by high performance liquid chromatography (HPLC).<ref name="ASBC">ASBC Methods of Analysis. Hops Method 14. Approved 1990. American Society of Brewing Chemists, St. Paul, MN, U.S.A. doi: 10.1094/ASBCMethod-Hops14</ref><ref name="Danenhower">Danenhower, T. M.; Force, L. J.; Peterson, K. J.; Betts, T. A.; Baker, G. A. HPLC Analysis of α- and β-Acids in Hops. J. Chem. Ed. 2008, 85(7), 954-956.</ref>
 =Materials and Methods=