Geochemical investigation of Vermont maple syrup in the context of terroir

Environmental Studies 360 Final Report Spring 2007

Environmental Studies Program Middlebury College

Middlebury, Vermont 05753

Dr. Molly S. Costanza-Robinson and the members of ENVS 360 (listed alphabetically)

Tripp Burwell, Anna Chavis, Nick Dickerson, Elizabeth Kelley, Adam Lovell

This report was written as part of a research project conducted under the supervision of Dr. Molly Costanza-Robinson for the Middlebury College course entitled “Environmental Science Practicum” (Environmental Studies 360). Copyright of this report and its contents is retained by the Trustees of Middlebury College. Please cite this report as follows: Costanza-Robinson, M.S.; Burwell, T.; Chavis, A.; Dickerson, N.; Kelley, E.; Lovell, A. 2007. Geochemical investigation of Vermont maple syrup in the context of terroir. Environmental Science Practicum research report: Environmental Studies Program, Middlebury College, Middlebury, Vermont. Additional copies of this report may be obtained as a pdf document accessible at http://www.middlebury.edu/academics/ump/majors/es/courses/service/ES360/, in hard copy form from the Program in Environmental Studies, Middlebury College, Middlebury, Vermont 05753 (802/443-5710) or by contacting Dr. Molly Costanza-Robinson (Department of Chemistry and Program in Environmental Studies, 802/443-5571, mcostanz@middlebury.edu).

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Table of Contents Abstract 1 Introduction 2 Terroir 2

Bedrock & Soil 3 Sap 5 Syrup Flavor 6 Methods 9 Overview 9

Hadley Sap 9 Sugarmaker Sap 12 Sap Processing and Characterization 12

Syrup Color Analysis 13 Alkyl Pyrazine Extraction and Analysis 13 Statistical Analysis 14 Results and Discussion 15 Hadley Samples 15 Sap Properties over the Collection Period 15

Correlations between Sap Properties 17 Changes in pH during Sap Processing 19 Syrup Color 20

Sugarmaker Samples 21 Sap Properties Across Bedrock Type 21

Correlations between Sap Properties 25 pH Trends during Sap Processing 29

Syrup Color 30 Alkyl Pyrazine Analysis 31

Conclusions & Future Work 33 Acknowledgments 35 Literature Cited 36

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Abstract

This study investigates the geochemical basis for terroir of Vermont maple syrup by

examining relationships between sap/syrup chemistry and the bedrock type underlying the

sugarbush. Sap samples were collected from 19 small-scale commercial sugarmakers overlying

limestone, schist, or shale bedrock; sap was also collected daily from the Hadley stand at

Middlebury College in order to compare tree-to-tree and day-to-day differences for a single

bedrock type (limestone). No significant differences were observed in °Brix, pH, electrical

conductivity, alkyl pyrazine (flavor compounds) content, or color of sap/syrup across bedrock

type (p > 0.05). In the Sugarmaker sap, °Brix and electrical conductivity were correlated (p <

0.01), as were °Brix and pH (p < 0.01); no correlation was observed in sap/syrup properties for

the Hadley samples. All but one syrup sample contained ethyl pyrazine in concentrations ranging

from 14.4-126.1 ng pyrazine/g syrup, although no significant correlation was found between

ethyl pyrazine concentration and bedrock type. Thus, bedrock type underlying the sugarbush

does not explain the observed differences in sap °Brix, pH, and electrical conductivity, nor could

an influence of bedrock on syrup color or alkyl pyrazine flavor compound development be

discerned.

Introduction

Maple syrup is an important part of Vermont’s culture and heritage. Maple sugaring is

one of the few agricultural practices native to the state of Vermont and not imported or adapted

from European agricultural practices. Sugaring was adopted by Vermonters from Native

Americans, although its exact origins are a matter of speculation and folklore.1 Vermont citizens

have recognized the value of the maple industry, and have actively supported the proliferation of

sugar maple (Acer saccharum) trees in the region. Other varieties of maple tree are also suited

for producing sap, including black maple (A. nigrum) and red maple (A. rubrum).2 Maple syrup

is an essential aspect of Vermont’s economy, generating about 15 million dollars annually in

direct sales.3 There are approximately 2000 known sugarers in the state, and countless more

small-scale private sugarmakers.4 Sugaring serves as a seasonal tourism draw and plays an

integral role in Vermont’s economy.5 Therefore, it is important to understand the potential

factors that influence the taste of maple syrup and how those factors might vary across the state.

Terroir

The word terroir is French for earth or soil, or the idea of taste of place. The term most

often refers to the complex interrelationships among climate, soil, geology, and other factors that

influence the character and quality of wine.6 The concept of terroir has been discussed in the

context of many food products, including cheese, cocoa beans, and chocolate.7 For instance,

flavors in the wine from the Seven Hills vineyard (Walla Walla Valley, WA) are influenced by

low humidity, long growing season, silty soil derived from Quaternary glacial sediments and

wind-blown loess overlying Miocene basalt, trellising methods, and the overall topographic

climatic setting of the vineyards.6 Microclimates within the Valley, combined with variations in

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soil and bedrock stratigraphy yield a range of growing conditions that influence grape

development. For example, good growing conditions can foster the growth of tap roots that can

access ground water, while poor soils can hinder grapevine water and nutrient uptake.

With the concept of terroir well established in relation to the flavor of wine, it is of

interest to investigate whether the concept can be applied to other food products, such as maple

syrup. Because the sap is influenced by the mineral uptake of the maple tree from its soil, it is

conceivable that the microhabitat of the tree ultimately influences the taste of the syrup. This

study borrowed the concept of wine terroir and evaluated relationships between bedrock and the

chemistry of the sap and syrup derived from the overlying trees. It was hypothesized that

bedrock directly influences the soil the tree is growing in, and indirectly influences the chemistry

of the sap produced by the tree, which, in turn, could ultimately influence the flavor of the syrup.

Bedrock and Soil

Soil formation is driven by interrelated factors, including time, topography, vegetation,

climate, and parent material. As bedrock weathers, it provides a source of minerals and nutrients

and becomes more conducive to plant growth, which, in turn, adds organic matter to the soil.

Bedrock and soils can play a role in terroir because these media influence the inorganic nutrients

that are taken up into the roots of plants. Studies have shown that due to differences in bedrock

types and soils, geographically adjacent vineyards can demonstrate quite different terroir even

though their major climatic features may be similar.6 For example, in the Holyoke range, an east-

west oriented basalt ridge in the Connecticut River Valley of Massachusetts, soil variables such

as pH and nutrients accounted for 51% of the variance in plant distribution.8 Furthermore, soil

not only influences different plant varieties in one location, but also influences one plant variety

3

in different locations. One study concluded that 48 samples of the same Chardonnay variety

grown in different areas of California exhibited different taste characteristics related to a

“regional uniqueness.”9

It is likewise hypothesized that sugar maple trees may also exemplify terroir of different

sugarbushes in various geographical locations in Vermont. Sugar maple stands occur principally

on three types of bedrock in Vermont: schist, limestone, and shale. Schist and shale exhibit

nearly identical elemental composition, because schist is simply the metamorphosed form of

shale. However, differences in the mineral composition may result in different mineral

solubilities and differing nutrient release to the soil. In contrast, limestone is chemically distinct

from schist and shale. Soils above limestone bedrock in VT have been shown to contain higher

concentrations of exchangeable calcium and magnesium than soils above schist or shale, while

soils atop schist and shale have more aluminum and potassium than limestone.10 These four

elements (Ca, Mg, Al, K) play an important role in grape development and, thereby, are

important for the production of a balanced wine.11 Magnesium is particularly important in wine

production because it assists in the uptake of other nutrients, aids the assembly of the chlorophyll

molecule, and develops a granular soil structure necessary for proper drainage and oxygenation.

However, excess magnesium can reduce the permeability for water and air in the soil, leading to

poor drainage.12 Thus, it is reasonable to assume that the chemical composition of the bedrock

and soil may influence sugar maple trees because of the differing soil elemental compositions,

water holding capacities, and pH.

4

Sap

The sugar in maple sap comes from photosynthetic products that are stored in the form of

starches within a tree over the course of the winter and converted to simpler sugars during the

following growing season.13 While mostly consisting of water, maple sap contains sugars in the

form of sucrose (98%), fructose and glucose (2%) and also contains myriad inorganic nutrients

(K, Ca, P, Mg, S), organic acids, amino acids, hormones, microbes, and ammonia.14 The amount

of sugar in the sap depends on various factors, including tree genetics, the quality of the site

where the tree grows (slope, aspect), the tree’s health and nutrition, diameter, size of the crown

relative to diameter, environmental conditions of the previous growing season, and the time of

year.15 For example, it has been suggested that trees with many branches and sun exposure

produce sap with high ˚Brix, that sap from open-grown trees have higher ˚Brix than forest-grown

ones, and that larger DBH and growth rates are all related to high sugar content. A single tree can

show variation in sugar content from season to season, but a consistent pattern has been observed

over multiple seasons that a “sweet tree” is always sweet relative to other trees. The sugar

content of individual trees and entire maple stands also varies through the season. Not only the

sugar, but the quantities and presence of all the different constituents of sap, such as amino acids,

vary among trees.16 For example, warmer air temperatures in Spring result in an increase in

amino acids that promote budding and contribute off-flavors to sap (e.g. buddy flavor).14

Maple sap is extracted from a tree following the winter months once the temperatures rise

above the freezing point during the day and fall below freezing at night.17 Unlike most trees,

maple tree fiber cells are gas-filled, not water-filled. This is important because once freezing

occurs, small ice crystals form inside each of these air-filled fiber cells, pulling moisture from

adjacent vessels and increasing the air bubble contraction. This pressure gradient pulls the sap

5

from the roots and into the branches as the gas in the fiber cells compresses and air dissolution

occurs. As the tree warms during the day, the wood fiber cells thaw and the gas comes out of

solution and expands, pushing the sap back down the tree. Maple sap can be harvested during the

thaw portion of the freeze-thaw cycle.

Syrup Flavor

Flavor is difficult to describe scientifically because there are hundreds of chemicals that

may contribute to a given taste,18 and in different concentrations and combinations, the same

chemicals can yield different flavors.19 The human factor adds additional complexity to the study

of flavor; people have different taste preferences and varying abilities to discern tastes. Caution

must be exercised when attempting to correlate flavor to specific concentrations of specific

flavor compounds because of the wide range of human odor and taste thresholds.20 This study

focused on the flavor effects of heterocyclic alkyl pyrazines, which are a family of pyrazine

molecules known for their nutty, roasted flavors (Figure 1).19

Figure 1. Structure of a pyrazine molecule.21

Alkyl pyrazines are created as a late-stage product of the Maillard reaction, a reaction

that is influenced by temperature, time, and pH as the sap is boiled into syrup.23 The Maillard

reaction is a critical pathway that contributes color and flavor to foods during high-temperature

processing, such as boiling, frying, and baking.23 The initial stage of the Maillard reaction

includes a condensation reaction between the amine group of free amino acids and the carbonyl

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group of the reducing sugars. This forms a Schiff base creating an alkaline environment. In this

stage, the sap remains essentially colorless. Next, the Amadori rearrangement occurs with

isomerization of the N-substituted glycosylamine, resulting in the transition from an aldose to a

ketose sugar derivative. In this stage, the sap begins to yellow and starts to have strong

absorption in the near-ultraviolet. Strecker degradation then occurs and a dicarbonyl is formed.

Although the exact mechanism has not been well characterized, the heterocyclic compounds

alkyl pyrazines are ultimately formed from these dicarbonyls. By this point, the sap has been

converted to syrup and has taken on the characteristic color for its quality grade.23

Methyl pyrazine, 2,6-dimethyl pyrazine, 2,3-dimethyl pyrazine, 2,5-dimethyl pyrazine,

ethyl pyrazine, and trimethyl pyrazine have been identified in maple syrup.24 While these alkyl

pyrazines are also found in non-maple syrups, genuine maple syrups have been shown to have

substantially higher quantities of total alkyl pyrazines than non-maple syrups.22 Trimethyl

pyrazine is present in maple syrups but only present in trace quantities in non-maple syrups,

suggesting that trimethyl pyrazine may be significant in imparting the characteristic maple

flavor.19 In one study, different grades of maple syrup exhibited significantly different quantities

of alkyl pyrazines: medium grade had the highest concentration of total pyrazines, amber grade

had the lowest, and light grade had a middling concentration.19 Across all grades, 2,6-dimethyl

pyrazine was found to be the alkyl pyrazine present in the highest concentrations.

The variable distribution of alkyl pyrazines might be explained by the different rates that

various amino acids react with two-, three-, and four-carbon fragments.16 The pH of sap may be

related to the distribution of alkyl pyrazines with more basic conditions favoring alkyl pyrazine

formation.22 Soon after boiling and evaporation begin, the sap becomes more alkaline due to the

formation of the Schiff base, but pH subsequently decreases until it is about neutral at the end of

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boiling.25 Little color is produced until after the sap reaches a pH of 9, when color increases

rapidly. It increases further as the concentration of the sap approaches that of finished syrup

(67.5 °Brix). During boiling, an induction period exists before alkyl pyrazines are observed,

attributed to the formation of precursors that interact to form alkyl pyrazines. After the induction

period, the concentration of alkyl pyrazines has been shown to increase linearly with boiling time.

Amber and medium amber syrups were preferred in one sensory evaluation study and were

found to contain higher levels of 2,3-dimethyl and 2-ethyl-3-methyl pyrazines but lower levels of

2,5-dimethyl pyrazine when compared with lighter syrups.30 The amber syrup, which was judged

to have the highest maple flavor, contained the lowest quantity of trimethyl pyrazine.

The specific flavors in various syrup grades likely depend on the initial sap chemical

composition, holding conditions, and processing methods.26 In this study, sap processing

methods were held constant in order to assess the potential influence of the type of bedrock

underlying the sugarbush. A relationship between syrup properties and bedrock underlying an

individual sugarbush has not been previously reported.

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Methods

Overview

This study investigated the concept of terroir through an analysis of relationships between

bedrock type, sap chemistry, and syrup alkyl pyrazine composition. Maple sap from 19 small-

scale Vermont sugarmakers overlying three types of bedrock (Figure 2) was characterized,

processed into syrup, and alkyl pyrazine profiles in the resulting syrup were explored. Sap and

syrup chemical properties including sugar content (°Brix), conductivity, pH, color, and percent

transmittance were determined. These samples are referred to herein as Sugarmaker sap/syrup. A

second group of samples, referred to herin as Hadley sap/syrup was used to assess tree-to-tree

and day-to-day variation in sap and syrup chemistry for trees located on a single bedrock type

(limestone). For this second group, five trees in the Hadley maple stand on the Middlebury

College campus in Middlebury, Vermont (Figure 3) were tapped. Finally, commercial syrup

samples were used to compare color, °Brix, and alkyl pyrazine content with Sugarmaker samples

and to verify analytical methods.

Hadley Sap

Five sugar maple trees were chosen in the Hadley stand, a mixed small forest near the

golf course at Middlebury College (Figure 3). Several criteria were used in choosing the trees,

including a healthy appearance, as much canopy exposure as possible, and a location less than 50

m from the cross country trail. All trees chosen were of similar diameter (30.9 – 37.1 cm at

breast height (147 cm)).

9

Figure 2. Simplified Vermont bedrock map showing locations of selected 2007 Sugarmakers. Only 19 participated in the study.

10

Figure 3. Photo showing Hadley stand on the Middlebury College campus and the five trees tapped for this study.

The Hadley maples were tapped on March 11th on their southern side (180º) at a height of

147 cm using a battery-powered drill. The holes (7/16 in.) were drilled approximately 4.8 cm

horizontally into the trunk, and the spiles were immediately placed in the fresh hole using a

rubber mallet. Spiles, buckets, and lids were all made from galvanized steel and contained no

lead. One bucket was hung from each spile and was checked every 24 hours for evidence of sap

flow. Sap was collected on ten viable days starting March 12th (eight of which were consecutive),

11

and refrigerated (5 °C) until processing. Hadley sap was processed within one week after

collection. If trees produced less than 600 mL of sap any given day, the sap was characterized

but was not processed into syrup.

Sugarmaker Sap

Twenty-four small-scale sugarmakers who did not blend their sap with other sugarers and

were located on a single bedrock type were selected. Samples were obtained from 19 of these

sugarmakers with maple stands situated on limestone (n = 5), shale (n = 5), schist (n = 8), and

granite (n = 1) bedrock, as shown in Figure 2. Sap samples were collected in glass 1 gal jugs at

the sugarhouse and immediately stored on ice in a cooler for transport. Sap samples were held in

the cooler between 1 and 12 h, depending on sugarbush location, before being refrigerated (5 °C).

Most Sugarmaker sap samples arrived just before and during Spring Recess (March 23 - April 2,

2007). Therefore, the processing of Sugarmaker sap usually occurred seven days later, but some

sap was stored for up to fourteen days.

Sap Processing and Characterization

Hadley and Sugarmaker saps were characterized and processed in an identical manner.

Sap was analyzed for conductivity (Curtin Matheson Scientific digital meter), pH (Orion 420 A+

Thermal Electron Corporation), and ºBrix (0-10% Sun Instruments low scale refractometer and

Brix 90 Reichert 50-90% Solids refractometer). The pH measurements were automatically

temperature corrected. Temperature was not recorded for all saps, which prevented manual

temperature correction of the conductivity measurements. Because saps were characterized

within minutes after retrieval from refrigerated storage, significant variation in sap temperature

was not expected. Thus, the conductivity values reported, although low due to the low

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temperature (~7 °C), can be compared across saps. The °Brix, a measurement of total dissolved

solids, and thus, an effective proxy for sugar content, was recorded to the nearest 0.1°.

The sap (600 mL) was boiled on a hot plate and magnetically stirred in a 1-L beaker. pH

and ºBrix were measured at 20-min intervals until the volume of liquid was reduced to 300 mL.

Sap was transferred to a 600-mL beaker, and measurements were continued at 20-min intervals.

When the sap volume was reduced to 150 mL, measurements were taken approximately every 10

min, and the sap was transferred to a 150-mL beaker and boiled until the volume was 40 mL. At

this point, the heat was reduced, and the liquid evaporated on low heat until it reached

approximately 67.5 ºBrix. Between each transfer, the sap was filtered through a coarse food-

grade paper filter to remove foam. The finished syrup was stored in glass vials and refrigerated

prior to color analysis and alkyl pyrazine extraction.

Syrup Color Analysis

To characterize syrup color and establish its grade, % Transmittance (% T) at 560 nm

was measured (Agilent 8453 Photodiode Array UV/Visible Spectrometer with UV-Visible

ChemStation software; 1-cm quartz cuvette). In addition to Sugarmaker and Hadley saps,

commercially graded syrups were also measured verify method accuracy.

Alkyl Pyrazine Extraction and Analysis

Alkyl pyrazine flavor compounds were extracted from 14 syrup samples using the

method of Akochi et al.22 Five syrups were not analyzed for alkyl pyrazine due to low syrup

volumes produced or errors during the extraction step. Syrup (approx. 15 g) was diluted with

deionized, organic-free water (100 mL) and stirred. The mixture was adjusted to pH 2 using

drop-wise addition of 11% HCl. NaCl (30 g) was added, and the mixture was stirred for 10 min.

13

Diethyl ether (150 mL) was added, the mixture was stirred for 30 min, and the ether layer was

decanted to waste. The aqueous phase was adjusted to pH 12 using drop-wise addition of 5 M

NaOH. Dichloromethane (20 mL × 5) was used to extract the alkyl pyrazines from the aqueous

phase.

The extracts were evaporated in a rotary evaporator at 40 °C to a volume of

approximately 2 mL, dried with magnesium sulfate, spiked with 100 ppm pyrazine internal

standard, filtered, and further evaporated to approximately 0.5 mL under a gentle stream of N2.

Extracts were diluted to 2 mL prior to analysis. GC-FID analysis was performed on an Agilent

6890N Network GC System with 5 μL autosampler injection on to a Supelcowax 10™ column

(30 m; 0.32 mm i.d.; 0.25 µm film thickness). The injector and detector temperatures were 200

ºC, and N2 was used as the carrier gas at 1 mL/min. The oven temperature program was as

follows: 40 ºC for 6 min, to 64 ºC at 1 ºC/min (hold 20 min), to 70 ºC at 1 ºC/min, and to 150 ºC

at 10 ºC/min. Alkyl pyrazine concentrations were quantified using the method of internal

standards. Pyrazine was used as the internal standard.

Statistical Analysis

Statistical analyses were performed on Microsoft® Excel 2003. Three-way Kruskal-

Wallis tests were used to examine differences in sap/syrup properties across the three main

bedrock types studied (limestone, shale, and schist), using the Winstat Excel add-in provided by

R. Fitch Software. Two-way two-tailed ANOVA tests were also used to compare grouped

shale+schist data to limestone data. Pearson linear correlations tests were used to examine 2-way

correlations between pH, conductivity, ºBrix, and sugarbush elevation. Granite was eliminated

from bedrock analyses due to its sample size of one, but was included in two-way correlations

between sap properties.

14

Results and Discussion

Hadley Samples

Sap Properties over the Collection Period

The sap from Hadley trees exhibited a similar pH trend over the collection period with

pH values ranging narrowly from 6.4 to 7.6 (Figure 4). Over the first three collection dates, all

the pH values rose. Following a four-day gap in the collection period when day/night

temperatures did not allow sap formation, pH rose again for all trees. Trees 1 and 2 peaked on

March 22nd, and Trees 3, 4, and 5 peaked on March 23rd. All pH values of sap exhibited little

variation with the exception of Tree 5 on March 22nd. Overall, the trends in sap pH are similar

for all trees in terms of their peaks, and fell within an expected range.14,22,27

66.26.46.66.8

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Figure 4. Hadley sap pH trends by tree over the collection period.

The sap from all Hadley trees also exhibited the same general pattern in ºBrix: rising

slowly, spiking to a peak on March 20th, and then dropping sharply for a day before gradually

15

leveling off (Figure 5). This pattern is reflective of the typical sap season.15 Average ºBrix during

the peak on March 20th was 4.58, and average ºBrix on the last date of the collection period,

March 25th, was 2.6. Sap ranged by 3.9 ºBrix over the entire collection period and among all

trees. Trees 4 and 5 tended to have the highest ºBrix, while Tree 3 had the lowest almost every

day. The ºBrix pattern is characteristic of the harvesting season, and the hypothesis that a “sweet

tree will always be a sweet tree” relative to others appeared to hold in this case.15

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Figure 5. Hadley sap °Brix trends by tree over the collection period.

All Hadley trees exhibited a similar trend in electrical conductivity (Figure 6). Sap

samples from Trees 5 and 3 tended to have high electrical conductivity while sap from

Tree 1 generally was the lowest, illustrating the consistent differences between individual trees

over the test period. Conductivity increased at the beginning of the run, displayed a decline after

three days starting on March 21st, and then slowly rose.

16

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Tree 1Tree 2Tree 3Tree 4Tree 5

Figure 6. Hadley sap electrical conductivity trends by tree over collection period.

Correlations Between Sap Properties

Correlations were explored between the three sap variables – ºBrix, conductivity, and pH

– over the collection period. No significant correlations were found (p > 0.05) (Figures 7-9). The

lack of significance between pH and conductivity may be due to myriad ions that influence

conductivity, but that may not affect pH. The correlations may have been significant had there

been more samples from more days, over several seasons, or from more trees. It is also possible

that no significant correlations exist between these three variables because of significant genetic

variation between individual trees.

17

200

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6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8

pH

Con

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(µm

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p > 0.05

Figure 7. Correlation between pH and conductivity in Hadley sap.

6.26.46.66.8

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r = -0.17p > 0.05

Figure 8. Correlation between pH and °Brix in Hadley sap.

18

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500

600

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1 2 3 4 5 6

ºBrix

Con

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r = 0.13p > 0.05

Figure 9. Correlation between electrical conductivity and °Brix in Hadley sap.

Changes in pH During Processing

Variation in °Brix and pH over the processing period was assessed in order to determine

patterns of change during processing, and to compare these changes to alkyl pyrazine formation.

The pH values started in a narrow range, fluctuated, and ended in a neutral range between 6.5

and 7.5 (Figure 10). The characteristic rise in pH in the beginning of processing was not seen for

all samples,22 perhaps due to confounding variables such as a long storage time before

processing or the presence of bacteria in the sap. Similarities do not appear for the same tree on

different days.

19

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Figure 10. Changes in pH during processing of Hadley sap.

Syrup Color

The Hadley syrups had % T placing it into dark grades: commercial, grade B, grade A

dark, and grade A medium (Table 1). The Hadley syrups did not deviate from the typical syrup

produced in the 2007 season, however. According to Henry Marckres, the Consumer Protection

Section Chief for the Vermont Agency of Agriculture, Food, and Markets, the syrup from the

2007 season has been dark, on the whole.29 He attributed this tendency to warm weather in

January, but does not know the exact cause of the darker color.

20

Table 1. Percent transmittance at 560 nm and Vermont grade of syrups for Hadley syrup.

Tree Collection Date % T at 560 nm VT grade 1 12-Mar 13.0 Commercial 2 12-Mar 0.1 Commercial 3 12-Mar 62.7 Grade A Dark 4 12-Mar 42.7 Grade B 5 12-Mar 47.6 Grade A Dark 4 13-Mar 73.3 Grade A Medium 2 14-Mar 38.9 Grade B 3 14-Mar 55.5 Grade A Dark 4 14-Mar 22.5 Commercial 4 20-Mar 70.8 Grade A Medium 5 21-Mar 74.7 Grade A Medium

Sugarmaker Samples Sap Properties Across Bedrock Type

Sap from 19 sugarmakers was characterized according to pH, °Brix, and conductivity and

these properties were compared across bedrock type (Figures 11-13). A Kruskal-Wallis three-

way test across bedrock type revealed no significant relationship between bedrock type and °Brix

(p = 0.66), conductivity (p = 0.97), and pH (p = 0.70). Because schist and shale have similar

elemental composition, saps from sugarbushes overlying these bedrocks were grouped and

compared to limestone saps using a two-way ANOVA test; differences among sap properties

between the two groups were statistically insignificant.

21

55.5

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77.5

88.5

9

Limestone Schist/Shale Schist Shale

Bedrock type

pH

n = 13 n = 5n = 8n = 5

3-way: p = 0.702-way: p = 0.75

Figure 11. Average pH for sap from trees above both schist/shale bedrock, schist, shale, and limestone bedrock (± 1 standard deviation)

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Limestone Schist/Shale Schist Shale

Bedrock type

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n = 13n = 5

n = 5n = 8

3-way: p = 0.662-way: p = 0.50

Figure 12. Average °Brix for sap from trees above both schist/shale bedrock, schist, shale, and limestone bedrock (± 1 standard deviation)

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duct

ivity

(µm

ho)

n = 13n = 5n = 8 n = 5

3-way: p = 0.972-way: p = 0.49

Figure 13. Average conductivity for sap from trees above both schist/shale bedrock, schist, shale, and limestone bedrock (± 1 standard deviation).

The average and total range in sap °Brix, pH, and conductivity for various groupings of

samples are shown in Table 2. The Hadley sap ºBrix and conductivity had a greater range than

the Sugarmaker samples, while the Sugarmaker samples showed a wider pH range than the

Hadley samples. The fact that the 5 Hadley trees showed greater variation in sugar content and

conductivity than composite Sugarmaker sap collected from hundreds to thousands of trees

demonstrates the important role that individual tree genetics can play in determining sap

chemistry, particularly if a small number of trees are considered. Variation across days of a

single (Hadley) tree over time suggests that sap samples received at different points in the season

will have different qualities independent of the sugarbush climate. Thus, differences in variables

such as ºBrix, pH, and conductivity across bedrock could be overshadowed by temporal and

genetic variation.

23

Sugarmaker schist saps showed the widest range for all three variables, while shale had

the narrowest range. The wide variation in sap properties from sugarbushes overlying schist

suggests that sap samples from one type of bedrock can exhibit significantly different properties.

Moreover, average elevation or variation in elevation across the bedrock types did not explain

the variation (Figure 14). For example, although shale showed the least variation in sap

properties, the elevation of the shale sugarbushes had the most variation and similar average

elevation to the schist sugarbushes.

Table 2. Average and range of sap properties for sap sample groupings.

°Brix pH Conductivity (μmho)

Sample Set Ave Range Ave Range Ave Range Hadley sap (n = 5) 3.1 3.9 7.01 1.15 496 477 Sugarmaker sap (n = 19)* 2.0 3.0 6.48 2.34 394 431

Schist (n = 8) 2.1 3.0 6.65 2.15 398 431 Shale (n = 5) 1.8 0.8 6.50 0.55 371 158 Schist/shale grouped (n = 13) 2.0 3.0 6.60 2.15 388 431 Limestone (n = 5) 2.1 1.7 6.33 0.77 409 266

* includes one granite sample

0200400600800

1000120014001600

Limestone Schist/Shale Schist Shale

Bedrock type

Elev

atio

n (m

)

n = 5 n = 13 n = 8 n = 5

Figure 14. Average elevation for sugarbushes above both schist/shale bedrock, schist, shale, and limestone bedrock (± 1 standard deviation).

24

Correlations Between Sap Properties °Brix and conductivity in the Sugarmaker sap samples were significantly and positively

correlated (r = 0.54, p < 0.01, Figure 15). However, this significance no longer holds when the

high-°Brix/high-conductivity schist sample is removed.

00.5

11.5

22.5

33.5

44.5

5

5 6 7 8 9

pH

°Brix

LimestoneSchistShaleGraniter = 0.63

p < 0.01

Figure 15. Correlation between Sugarmaker sap °Brix and conductivity.

The correlation between pH and °Brix was also found to be significant (r = 0.63, p < 0.01,

Figure 16). Higher soil pH can result from basic cations in the soil solution (Ca2+, Mg2+, K+),

which are required for nutrient production in trees. Therefore, greater nutrient access may result

in more sugar production. However, the significance of the °Brix-pH correlation no longer holds

when the high-°Brix/high-pH schist sample (same outlier as in Figure 15) is removed. The

correlation between conductivity and pH in the Sugarmaker samples was positive but not

significant (r = 0.28, p > 0.01, Figure 17).

25

00.5

11.5

22.5

33.5

44.5

5

5 6 7 8 9

pH

°Brix

LimestoneSchistShaleGraniter = 0.63

p < 0.01

Figure 16. Correlation between Sugarmaker sap pH and °Brix.

0

100

200

300

400

500

600

700

5 6 7 8 9

pH

Con

duct

ivity

(µm

ho)

LimestoneSchistShaleGraniter = 0.28

p > 0.10

Figure 17. Correlation between Sugarmaker sap conductivity and pH.

26

Neither °Brix (Figure 18) nor conductivity (Figure 19) of the Sugarmaker sap was

correlated to sugarbush elevation (p > 0.10); however, sap pH was positively correlated with

elevation (p < 0.05, Figure 20). Soil pH and elevation would be expected to be negatively

correlated (i.e., more acidic at higher elevations), indicating that the tree itself may control sap

pH more than the soil.

00.5

11.5

22.5

33.5

44.5

5

0 500 1000 1500 2000

Elevation (m)

°Brix

LimestoneSchistShaleGranite

r = 0.10p > 0.10

Figure 18. Correlation between Sugarmaker sap °Brix and sugarbush elevation.

27

200250300350400450500550600650700

0 500 1000 1500 2000

Elevation (m)

Con

duct

ivity

(um

ho)

LimestoneSchistShaleGranite

r = 0.08p > 0.10

Figure 19. Correlation between Sugarmaker sap conductivity and sugarbush elevation.

5

5.5

6

6.5

7

7.5

8

8.5

0 500 1000 1500 2000

Elevation (m)

pH

LimestoneSchistShaleGranite

r = 0.39p < 0.05

Figure 20. Correlation between Sugarmaker sap pH and sugarbush elevation.

28

pH Trends During Processing

The pH and °Brix variation in trends over processing time were measured to assess

whether there were any differences among bedrock types with regard to chemical changes during

processing. Moreover, changes in pH influence the Maillard reaction that is at least partially

responsible for flavor development in maple syrup.23 Thus, pH trends were important to examine

for saps across bedrock types. Individual saps do not appear to follow a set trend in pH trend

over time, although, across all samples pH is seen to rise and then fall during processing (Figure

21). Limestone syrups tended to finish with pH values between 6 and 7, while schist/shale

samples exhibited greater variation in finished pH.

55.5

66.5

77.5

88.5

99.5

0 50 100 150 200

Time (min)

pH

Limestoneschist and shale

Figure 21. Changes of pH values over time for Sugarmaker samples in relation to bedrock type.

29

Syrup Color

Percent transmittance (% T), a measure of syrup grade, was not significantly different

across the three bedrock types (p = 0.40). Measured % T placed many of the samples in dark

syrup grades, but absolute % T values showed considerable variation between samples (Table 3).

The % T for commercial syrups was also measured to verify the analysis method. Measured % T

at 560 nm fell within specified ranges for each commercial color standard, except for the Grade

A dark commercial sample, which had lower % T than the industry standard for that grade

(Table 4).

Table 3. Sugarmaker syrup %T at 560 nm and associated Vermont grade.

Bedrock %T at 560 nm VT Grade Limestone 50.54 Grade A Dark Limestone 17.46 Commercial Limestone 6.60 Commercial Limestone 5.71 Commercial Limestone 3.68 Commercial Schist 59.93 Grade A Dark Schist 19.59 Commercial Schist 8.45 Commercial Schist 6.21 Commercial Schist 5.05 Commercial Schist 1.59 Commercial Schist 0.74 Commercial Schist 0.07 Commercial Shale 8.64 Commercial Shale 7.58 Commercial Shale 2.97 Commercial Shale 2.56 Commercial Shale 1.68 Commercial

30

Table 4. Measured and industry standard % T at 560 nm for commercial syrup samples.

VT Grade Measured % T at 560 nm Industry Standard %T at 560 nm Grade A Medium 65.6 60.5-74.9

Grade A Dark 40.0 44.0-60.4 Grade B 37.9 27.0-43.9

Commercial 13.1 <27

Alkyl Pyrazine Analysis

None of the samples were found to have 2,6-dimethyl, 2,3-dimethyl, trimethyl, or 2-

ethyl-3-methyl pyrazines. Only one sample had 2,5-dimethyl pyrazine (160.8 ng/g syrup), and

one was the only sample to have methyl pyrazine (2.4 ng g ). Ethyl pyrazine was observed in all

but one sample and ranged between 14.4 ng/g and 126.1 ng/g (Figure 22).

020406080

100120140160

Limestone Schist/Shale Schist Shale

Bedrock type

ng E

tP/g

syr

up

n = 5 n = 13 n = 8 n = 5

Figure 22. Concentrations of ethyl pyrazine (ng/g of syrup) across limestone bedrock, schist/shale, schist, and shale bedrocks (± 1 standard deviation)

The measured alkyl pyrazine content differed considerably from literature reports. Alli et

al.24 reported the presence of many more alkyl pyrazine compounds in maple syrup samples,

including methyl-, 2,6-dimethyl, 2,3-dimethyl, 2,5-dimethyl, ethyl, and trimethyl pyrazines in

31

maple syrup. Ethyl pyrazine was the ubiquitous pyrazine in the current study, but, of those

quantified by Alli et al., ethyl pyrazine was found at the lowest concentrations. Akochi et al.30

also found that 2,6-dimethyl pyrazine was the alkyl pyrazine present in the highest

concentrations in light, medium, and amber syrups, while it was not observed in the current

samples. However, Akochi et al. only found 2,6-dimethyl after 120 minutes of boiling, and most

samples in the current study were heated for approximately 120 minutes, but actually boiled for

less than that. Finally, the use of only 600 mL of sap (~15 g syrup) in this study could have

hindered the alkyl pyrazine analysis because of analytical detection limits.

32

Conclusions & Future Work

A significant relationship was found between °Brix and conductivity and between pH and

°Brix in the Sugarmaker samples. No significant correlations were found, however, between

°Brix, conductivity, or pH of sap samples and underlying bedrock types. Very few alkyl

pyrazines were found in the Sugarmaker sap samples; no 2,3-dimethyl-, 2-ethyl-3-methyl-, and

trimethyl pyrazines were found. All but one syrup sample contained ethyl pyrazine, one sample

had 2,5 dimethyl pyrazine, and two had methyl pyrazine. However, no significant correlation

was found between ethyl pyrazine profiles in syrup samples and bedrock types. The lack of alkyl

pyrazines may have been a result of the short boiling time when processing the sap or to the

extraction of a small quantity of syrup. Further verification of the extraction method should also

be conducted.

The results of the current study are consistent many previous findings, such as seasonal

and individual patterns and variation in sap sugar content, yet do not provide new definitive

findings in terms of bedrock and terroir. Given our data, it is not possible to assert that

sugarbushes on one bedrock type show greater quantities or types of alkyl pyrazines than

sugarbushes on a different bedrock type. This study, however, does not eliminate the possible

influence of local factors on the taste of maple syrup or the possibility that microclimatic factors

play a role in the taste of this important Vermont gourmet product.

Further work should continue to focus on the investigation of differences in sap and syrup

in relation to bedrock, but also should explore impacts of elevation of the sugarbush. In addition

to alkyl pyrazine profiles, elemental or amino acid profiles could also be analyzed. Rather than

analyzing syrup samples that were processed in the lab, syrup samples could be taken from

sugarmakers and analyzed, taking into account the variance in processing methods and using the

33

differing commercial methods (plastic tubing, reverse osmosis) as one set of variables. This

could allow for more time to be spent on extraction as opposed to processing, which is important

because a larger number of samples in each bedrock is needed for more statistically significant

results and conclusions to be drawn. Combining analysis of finished syrup with initial sap from

the sugarmakers might allow some measure of processing effects on syrup. Further method

development and verification should be applied to the alkyl pyrazine extraction process to assure

that the reason alkyl pyrazines were not detected in the syrup was not due to the fact that they

were lost in the extraction process or that the extraction process was not capturing all the alkyl

pyrazines.

Tree growth and aging could be a significant factor in the production of maple sap.

Further studies should examine the effect of the rate of tree growth on ºBrix, pH, and

conductivity of sap. Tree development and health could have an impact on the amount of sugar

devoted to off-season growth or sap production. Tree age could also be expected to influence the

concentrations of both amino acids and sugars in maple sap, especially given the observed

variation between the °Brix, pH, and conductivity in individual trees in a single sugarbush.

Exploring differences in sugarbushes and their production of different types and quantities of

sugars and amino acids would be important in assessing the resulting flavor compounds and their

link to the soil and climatic environment of the tree.

34

Acknowledgements Thank you to the twenty-one Vermont sugarmakers for contributing to the success of this

research project, to Carolyn Barnwell for coordinating the project and for collecting and storing

sap from Sugarmakers, to Lee Corbett and Professor Jeff Munroe for their help in background

knowledge, and to Professor John Elder for the tour of his sugarbush and sugar house.

35

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