Infrared spectral analysis of sugar profiles of worts from varying grist to liquor ratios using infusion and ramping mash styles

Journal of the Institute of BrewingVolume 122, Issue 3 p. 437-445 Research articleFree Access Infrared spectral analysis of sugar profiles of worts from varying grist to liquor ratios using infusion and ramping mash styles Glen Fox, Corresponding Author Glen Fox Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Centre for Nutrition and Food Science, Leslie research Centre, Toowoomba, Qld, AustraliaCorrespondence to: G. Fox, Queensland Alliance for Agriculture & Food Innovation, The University of Queensland, Centre for Nutrition and Food Science, Leslie research Centre, Toowoomba, Qld 4072 Australia. E-mail: [email protected]Search for more papers by this author Glen Fox, Corresponding Author Glen Fox Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Centre for Nutrition and Food Science, Leslie research Centre, Toowoomba, Qld, AustraliaCorrespondence to: G. Fox, Queensland Alliance for Agriculture & Food Innovation, The University of Queensland, Centre for Nutrition and Food Science, Leslie research Centre, Toowoomba, Qld 4072 Australia. E-mail: [email protected]Search for more papers by this author First published: 25 July 2016 https://doi.org/10.1002/jib.341Citations: 18AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Fermentability is an important trait for the brewing industry. Current industry methods lack the predictive capacity to accurately estimate how well a wort will ferment in the brewhouse. Wort from two mashing styles (high-temperature infusion and low-temperature ramping), and under varying grist to liquor (G:L) ratios, were produced and the differences in maltose, maltotriose and glucose were measured. The two mashing styles showed differences in original extract (Plato) values between the G:L ratios with a 1:2 G:L having the highest original extract. Maltose was the most abundant sugar in all sample types. All worts were scanned using Fourier transform infrared (FTIR) and the spectra also showed differences between the original extract and final extract with most of the changes around the spectral region associated with carbohydrates, a result of sugar utilization by the yeast. FTIR calibrations for extract and fermentable sugars all had r2 values >0.98, with ratio of standard error of prediction to standard deviation >5. The results indicated changing fermentable sugar levels, thus making a prediction of fermentability possible. The FTIR also provided a rapid measure of changes in the fermentable sugar profile, which could assist maltsters and brewers in monitoring malt and beer quality. Copyright © 2016 The Institute of Brewing & Distilling Introduction The malting and brewing industry assesses the quality of malt using a number of parameters. Hot water extract (HWE), expressed as a percentage of soluble solids or as original extract (OE) is one of those parameters. It provides information on the amount of soluble material produced in the wort during mashing. These solutes include sugars, amino acids, small proteins, minerals, vitamins and water-soluble phenolics 1. However, a number of variables influence the extract, including the malt quality (level of modification and the enzymatic potential of the malt), the style of mashing (low-temperature ramping vs. high-temperature infusion), the ratio of malt to water (grist: liquor) and the style of beer being produced. Each of these variables have been shown to have an impact on the level of extract 2, 3 and for components of the extract 4, 5, with the latter being important for fermentation. Several industry methods are routinely used to measure extract. The European Brewery Convention (EBC) method or the similar American Society of Brewing Chemists congress method, which use a ramping temperature during mashing, are commonly used to assess malt in breeding programs as these are the methods industry uses to evaluate new varieties. Globally, the more common commercial mashing style is the high-temperature infusion (~65 °C) for a minimum of 60 min. The EBC style is suited to enzyme survival whereas the latter quickly gelatinizes starch and enzyme survival is time limited to usually <30 min. Industry standardized methods routinely report the extract as an HWE percentage. Nevertheless, the Plato value is calculated and this value is more relevant to brewers. In addition, these standard methods will result in a Plato value <9, mostly owing to the grist: liquor (G:L) ratio, but brewers are more interested in extracts with a Plato value >10 and for high-gravity brewing (lower G:L ratio) a Plato value >13. Fermentability is of equal importance to extract, and like extract, the fermentation has a number of variables that affect the final amount of alcohol produced, flavours, aromas and overall beer quality 3. In addition, the strain of yeast is critical, as it transforms the profile of the wort components leaving unfermentable material, such as higher-order sugars, some of which influence final beer quality. However, there is a preferred profile of fermentable sugars by yeast strains. This profile of sugars in the wort comes from barley starch and some non-starch polysaccharides 6. Barley starch comprises two polymers, amylopectin and amylose, which are thousands of glucose units linked by α-1,4 bonds into chains, with amylopectin being branched with α-1,6 bonds. During mashing these polymers are degraded by starch-degrading enzymes. These enzymes, known as diastase enzymes (α-amylase, β-amylase, limit dextrinase and α-glucosidase) degrade starch into small sugar units, which are solubilized in the wort. Maltose is the most abundant sugar but glucose, maltotriose and maltotetrose, higher-order oligosaccharides and dextrins are present, although most brewing yeasts preferentially consume maltose before the larger sugar units. The action of these enzymes in producing the sugar profile is controlled partly by the style of mashing but also grist size and G:L ratio 7. Several studies have attempted to predict fermentability using a number of malt parameters and measures of individual sugars 3, 8, 9. Over recent decades, barley breeders have released varieties with higher levels of these diastase enzymes. Thermostable isoforms of β-amylase have been identified and introgressed into new varieties 7. The level and structural complexity of the starch substrates, amylopectin and amylose have been ignored, in terms of understanding if any structural variations of these polymers have an effect on the rate of diastase enzyme action and the resultant fermentable sugars. Amylopectin is the most abundant individual polymer in barley but there has been limited research into understanding the complex structure of the largest single component in barley (>30% of total grain composition). The structure of amylopectin and amylose are broadly known, with amylopectin likened to a highly branched 'tree', and amylose, like a very tall tree with few branches. Only recently has the fine structure in barley been started to be elucidated in terms of variations in chain lengths of amylose and amylopectin impacting the fermentable sugar profile 6. Variations in the level of amylopectin branching and the chain lengths of amylopectin and amylose in barley and malt are affected by genotype and environment 10. However, there has yet to be a study showing any relationship between variations in starch chain length and fermentable sugar profile. The production of wort and its subsequent analysis is time consuming so less expensive and rapid predictive methods have been utilized. Near-infrared spectroscopy has been used for decades to predict barley malt traits associated with improved quality 11 including in single grains of barley 11-13 and starch, proteins and lipids in barley grain, malt and wort 14. Near-infrared spectroscopy has been used to predict malt and wort quality 15, 16 and beer quality 17, 18 but its use in breeding programmes is where is has the greatest value 19. While mid infrared technology (IR) has had limited used in plant breeding selection 20, it has been applied to investigate wort quality 21 and, fermentation and wort sugars 22. It has also been used to monitor the changes in the barley during malting 23. While both the near-infrared and IR offer excellent platforms for the rapid assessment of multiple components within a wort, the IR spectra region from 2500 to 4000 nm is associated more with individual chemical bonding. This investigation was designed to study the fermentable sugar profile produced from two mashing experiments with the resultant worts analysed for glucose, maltose and maltotriose, before and after fermentation. The first experiment was to assess the fermentable sugar profiles of malts with differing levels of modification using two different mashing styles (infusion and ramping) with a fine and coarse grist. For the second experiment, differing G:L ratios were studied to assess any possible changes in the sugar profiles owing to possible enzyme protection from the higher grist ratios, but again comparing the two different mashing styles (infusion and ramping). Finally, worts from both experiments were scanned in a Fourier transform infrared (FTIR) instrument to assess the possibility of calibration development and understanding the spectral associations with fermentable sugars from each mash. Materials and methods Commercial malt production Commander is an accredited Australian malting variety for domestic and export brewing. It has the low thermostability β-amylase allele (Sd1). The barley was malted in a Joe White automatic micromalting unit at the Cargill malt facilities in Adelaide. Standard malt analysis was performed according to EBC methods 24 (Table 1). Table 1. EBC analysis of malt malted in the presence (+) or absence (−) of gibberellic acid (GA) Low KI (−GA) High KI (+GA) Difference between high and low KI malts Clarity of wort 1 1 Extract fine grind (DB, %) 79.3 80.5 1.2 Colour of wort (°EBC) 2.8 4.1 1.3 Diastatic power (DB, WK) 372 481 +109 Free amino nitrogen (FAN, mg/L) 132 187 +55 Total protein (DB, %) 10 9.9 Soluble protein (DB, %) 3.44 4.48 +1.04 Kolbach index (KI) 34.4 45.3 Friability (%) 79.2 86.6 Viscosity (mPa s) 1.51 1.47 −0.04 Wort β-glucan (mg/L) 202 93 −109 Saccharification time (min) 15 10 −GA no addition of gibberellic acid; +GA addition of gibberellic acid (0.4 ppm); DB, dry basis; WK, Windisch Kolbach. Grist and wort production Grists were prepared by milling malt in a Buhler Miag disc mill. Two grist sizes were milled, a coarse (1.0 mm) and a fine (0.2 mm). Experiment 1. EBC and Institute of Brewing standard mashes EBC (ramping temperature) method The EBC wort was prepared following the EBC standard hot water extract method 24. For this method, 50 g of milled malt was mashed in 200 mL warm liquor (45 °C) for 30 min with constant stirring. At 30 min, the program automatically ramped up to 70 °C (in 30 min). At this point, 100 mL of hot water (70 °C) was added and the mash remained at the temperature for 60 min with constant stirring. The mashes were cooled to room temperature and made up to 450 g with room temperature water prior to filtration. The wort was filtered through Whatman no. 597 1/2 filter paper, with the first 100 mL of wort returned. To obtain sweet wort samples for subsequent FTIR and fermentable sugar analysis, 10 mL was subsampled after the return of the first 100 mL. Specific gravity was measured on 10 mL of wort in a DMA 45 (Anton Paar, Germany). The specific gravity was used to calculate HWE and original extract (degrees Plato, °P). Institute of Brewing (high-temperature infusion) method The Institute of Brewing (IoB) method was conducted following the standard IoB method 25 with 50 g grist in 360 mL hot liquor (65 °C), mashed with constant stirring for 60 min. The worts were cooled to room temperature and made up to 450 g with room temperature water prior to filtration. The final G:L ratio was 1:8 prior to filtration. Filtration was carried out as described for the EBC method above. Subsampling for specific gravity, FTIR analysis and fermentable sugar analysis was carried out as described for the EBC method above. Experiment 2. Variation in G:L ratios using EBC and IoB mashes For experiment 2, the IoB and EBC methods were followed as described above with a minor variation to the IoB method where only 180 mL was added as the first water addition and an additional 180 mL was added at 30 min into the mash. The G:L ratios were 1:2, 1:3 and 1:4. For the IoB and EBC mashes, all combinations of malt modification (n = 2), grist size (n = 2) and G:L ratio (n = 3) were used. These were performed in duplicate and the total number of mashes for each mashing method was 24. However, HWE was not calculated owing to the differences in the G:L ratio used in the standard methods above, for example with the 1:2 G:L ratio, the HWE would be >100%, so extract levels were reported as OE (°P). Subsampling for FTIR and fermentable sugar analysis and specific gravity was carried out as described for Experiment 1 above. Fermentation For both experiments, the same fermentation method was conducted. Filtered worts were boiled in 500 mL Schott bottles for 10 min. The boiled worts were cooled to room temperature and topped up to initial weights with boiled (and cooled) distilled water. Three grams of Mauribrew Lager 497 yeast (Mauribrew, Toowoomba, Australia) was added to ensure full and rapid fermentation (less than 24 h). The Schott bottles were then sealed with a screw cap fitted with a U-tube containing sterile water to allow CO2 escape, and placed on an orbital rocker (120 oscillations per min) for 24 h at room temperature (21 °C). After 24 h, 10 mL of the fermented wort was centrifuged at 4000 g for 10 min and density was measured as described above. Final apparent attenuation limit (AAL) and apparent extract (AE), expressed as °P, were then calculated. HPLC Levels of glucose, maltose and maltotriose in sweet and fermented worts from the above experiments were determined using high pressure liquid chromatography (Agilent 1100; Agilent Technologies, Waldbronn D-76337, Germany) with a Prevail Carbohydrate ES 5 μm column (250 × 4.6 mm; Grace Davison Discovery Sciences, Australia), using an isocratic mobile phase at a flow rate of 1.0 mL/min. Standards of glucose, maltose and maltotriose were prepared at concentrations expected as per Experiment 1. For the 1:2 OE wort from experiment 2, the wort was diluted 1:20. FTIR A drop of each filtered wort sample, was placed on the diamond surface of an attenuated total reflectance (ATR) Perkin Elmer 100 FTIR instrument. Standard solutions of maltose and glucose standards, each 10% (w/v), were also scanned. The spectrum for each sample was obtained by taking the average of 32 scans (resolution of 4 cm−1, between 4000 and 850 cm−1) with a scanner velocity of 7.5 kHz (background of 64 scans). The ATR diamond surface was cleaned with ethanol (95% v/v) before each sample was scanned. The spectra were collected using Perkin-Elmer Spectrum software. Multivariate data analysis FTIR spectral data, along with calculated OE and AE (°P) values and sugar results, were analysed using Unscrambler (X3 Camo, Norway) for chemometric analysis. Principal component analysis (PCA) was performed on the spectra to examine the grouping of samples and to identify possible outliers. The raw spectra were processed using standard normal variate (SNV) with first derivative Savitzky–Golay transformation with 19 smoothing points. For calibration development, the same math treatments used for PCA were used to build a partial least squares regression. The calibrations were developed using all spectra. A full cross-validation (leave one out) option was used as the validation method. The ratio of standard deviation to standard error of prediction (RPD) was used to consider the potential application of each model, where an RPD >3 could be used in quality assurance 26. Results and discussion Experiment 1. Comparison between EBC and IoB extract results The EBC results (Table 2) showed similar extract results for the EBC mash to the data supplied with the malt (Table 1), indicating consistency in the use of the EBC industry method between two laboratories. From experiment 1, the EBC mash produced slightly higher HWE (%) and OE (°P) than the IoB mash, indicating a higher level of soluble solids. This is expected as the mash style allows for each enzyme group, glucanase, proteases, and amylases and limit dextrinase, to be highly active in degrading their specific substrates. The fine and coarse grist difference was higher for the low Kolbach index (KI) with the EBC and IoB methods. Table 2. European Brewery Convention (EBC) and Institute of Brewing (IoB) results using the standard industry methods (experiment 1) Mashing style Kolbach level (KI) Grist Hot water extract (%) OE F/C difference Apparent attenuation limit (%) OE (°P) AE (°P) OE maltose (%) OE maltotriose (%) OE glucose (%) AE maltose (%) AE maltotriose (%) AE glucose (%) EBC High Coarse 77.9 82.9 8.52 1.51 6.9 1.4 1.2 a b a EBC High Fine 80.1 0.2 85.3 8.73 1.33 4.5 1.0 0.8 a b a EBC Low Coarse 76.6 81.5 8.39 1.61 5.7 1.1 0.7 a b a EBC Low Fine 79.2 2.6 80.7 8.64 1.70 5.3 1.2 0.8 a a a IoB High Coarse 78.8 86.8 8.60 1.15 4.8 0.9 0.5 0.1 b a IoB High Fine 79.6 0.8 86.9 8.69 1.17 4.8 1.0 0.6 a b a IoB Low Coarse 76.9 86.7 8.42 1.15 5.0 0.9 0.5 a a a IoB Low Fine 79.1 2.2 85.3 8.64 1.29 4.7 0.9 0.5 a a a F/C, Difference in original extract between fine and coarse grists; OE, original extract (degrees Plato) as water and soluble solids; AE, apparent extract (degrees Plato) as water, unfermented solids and alcohol; a, no peak visible; b, peak visible but <0.1%. Table 2 shows the results for AAL and there was >80% fermentability for all combinations of mashing styles, KI levels and grist sizes. The highest levels of AAL were for the high KI, fine grist malt in both mashing styles. The IoB mash also had the highest level of AAL for both KI malts and both grist sizes indicating a greater change in the extract solids than for the EBC method. The level of maltose, maltotriose and glucose was either <0.1% or not detectable in the AAL from all samples, suggesting a high level of utilization of these fermentable sugars by the yeast in the 24 h fermentation period. Experiment 2. Varying G:L ratios in EBC and IoB mashes Table 3 shows the original and apparent extract for each mashing style and compares G:L ratios. For 1:2 G:L, the EBC original extract was higher than the IoB OE from both the high and low KI malt, and the coarse and fine grist. For the IoB, the high KI fine grist was slightly higher than the coarse grist. A similar effect was observed for the AE as for the QE. The difference between the OE and AE was slightly higher for the EBC high KI than the IoB but the low KI was similar between mashing style. The EBC mashing style offers a better environment for all three enzyme groups, glucanase, proteinase and starch degrading enzymes to degrade their respective substrates owing to the lower mash-in temperatures and slow ramp to 70 °C. The finer particle size also provides easier access to the substrates for the enzymes. Table 3. Results for original extract and apparent extract expressed as Plato, and sugar levels (experiment 2) Mashing style Kolbach level (KI) Grist Original extract (°P) Apparent extract (°P) Change between OE and AE (°P) Difference between OE and AE (%) OE maltose (%) OE maltotriose (%) OE glucose (%) AE maltose (%) AE maltotriose (%) AE glucose (%) G:L 1:2 EBC High Coarse 15.50 2.16 13.34 86.06 8.8 1.5 1.5 0.35 0.1 a EBC High Fine 15.90 2.27 13.63 85.72 7.3 1.3 0.8 a b a EBC Low Coarse 15.55 2.87 12.68 81.54 8.7 3.8 2.9 a b a EBC Low Fine 15.80 2.99 12.82 81.11 7.8 1.8 1.1 b a a IoB High Coarse 14.15 2.13 12.02 84.96 8.1 1.2 1.6 0.17 0.16 a IoB High Fine 15.00 1.81 13.19 87.93 7.4 1.4 1.3 a 0.12 a IoB Low Coarse 14.25 2.61 11.65 81.72 7.9 1.3 1.2 a 0.13 a IoB Low Fine 14.25 2.52 11.74 82.35 8.4 1.4 1.2 a 0.13 a G:L 1:3 EBC High Coarse 10.20 1.60 8.60 84.31 6.0 1.4 1.0 a b a EBC High Fine 12.90 1.73 11.18 86.15 5.7 1.1 0.9 a b a EBC Low Coarse 10.20 1.96 8.24 80.78 5.7 1.2 0.8 a a a EBC Low Fine 10.25 2.01 8.25 80.44 5.7 1.1 0.8 0.12 0.22 a IoB High Coarse 10.15 1.27 8.89 87.54 7.1 1.2 1.1 0.13 0.13 a IoB High Fine 10.90 1.35 9.55 87.62 5.6 1.1 0.8 0.14 0.15 a IoB Low Coarse 9.70 1.54 8.16 84.12 7.4 1.2 0.9 a b a IoB Low Fine 10.05 1.61 8.44 83.98 6.6 1.0 0.8 a b a G:L 1:4 EBC High Coarse 8.10 1.50 6.61 81.67 5.3 1.2 0.8 a b a EBC High Fine 7.65 1.38 6.27 81.97 4.5 1.0 0.8 a b a EBC Low Coarse 7.40 1.50 5.91 79.80 4.1 0.8 0.6 a b a EBC Low Fine 7.65 1.54 6.11 79.87 3.3 0.8 0.5 b b a IoB High Coarse 7.50 0.91 6.59 87.87 4.2 1.0 0.7 b b a IoB High Fine 7.70 1.05 6.66 86.43 4.1 0.9 0.6 0.26 0.17 a IoB Low Coarse 7.35 1.26 6.09 82.86 4.5 1.1 0.6 0.14 0.13 a IoB Low Fine 7.50 1.21 6.30 83.93 4.3 0.9 0.6 a b a OE, Original extract (degrees Plato) as water and soluble solids; AE, apparent extract (degrees Plato) as water, unfermented solids and alcohol; a, no peak visible; b, peak visible but <0.1% The OE values would be considered typical for these industry methods. There were similar OE values for both mash styles and at each G:L ratio. However, there were differences in AE values between mashing styles and G:L ratios. The high KI malt had a higher AE than the low KI malts, suggesting there was more fermentable material in the wort. There was also a greater difference in the AE between the high and low KI malt samples than for the 1:2 G:L ratio of those malts. The 1:4 G:L ratio had a much lower OE and AE compared to the two other G:L ratio samples. The low KI malt from the EBC mash had very low AE values, even when compared with the IoB style for the low KI malt. The IoB style showed similar values in the difference between OE and AE (an indication of fermentability), suggesting a consistent action by starch degrading enzymes across the three G:L ratios. The 1:2 G:L mash from both styles generally had a higher level of maltotriose in the original extract than the other two G:L ratio mashes. The residual maltotriose was also high in the AE for the 1:2 ratio as well. Correlations between extract and fermentable sugars A correlation analysis between all G:L ratios, both mash styles with both KI malts and fermentable sugars showed that maltose was highly correlated to OE (r2 = 0.6823; Table 4). Glucose and maltotriose were also highly positively correlated to OE. When considering the concentration of maltose in the extract, it was highly correlated to the AE (r2 = 0.4998) using the rapid, fermentation method. This data supports the well-known composition of wort, with maltose as the most abundant fermentable sugar, owing to the action of β-amylase on the reducing end of the starch chains. There was also a positive relationship between glucose and OE as well as maltotriose and OE. This was also the case with glucose, maltotriose and AE. Table 4. Coefficient of determination values between original extract, apparent extract and fermentable sugars from both mash styles, all G:L ratios data combined OE AE OE_G OE_M OE_Mtri OE 1.0000 AE 0.6762 1.0000 OE G 0.4842 0.4462 1.0000 OE M 0.6823 0.4998 0.6143 1.0000 OE Mtri 0.3415 0.4461 0.8010 0.3460 1.0000 OE, Original extract (degrees Plato) as water and soluble solids; AE, apparent extract (degrees Plato) as water, unfermented solids and alcohol; OE_G, level of glucose in original extract; OE_M, level of maltose in original extract; OE_Mtri, level of maltotriose in original extract. Infrared spectral analysis The above results describe the structure and composition of worts scanned in an effort to build a FTIR model for extract and fermentable sugars. Figure 1 shows the spectra of all the original extract samples (Fig.1a), fermented extract samples (Fig. 1b), and the average of OE (red) and AE (blue) samples (Fig. 1c). The spectra for the OE and AE are dominated by the water peaks 3300 and 1600 cm−1. In Fig. 1(c), when comparing the average of the OE (red) to the average spectra of AE (blue), there is a large noticeable difference in spectra from 1200 to 950 cm−1. This spectral region is associated with carbohydrates and the change in spectra between OE and AE in this region is associated with changes in sugars. One of the major differences between OE and AE (fermented wort) is the change in the sugars where there is reduction in fermentable sugars, specifically maltose, which is usually preferentially consumed by yeast during fermentation. Figure 1Open in figure viewerPowerPoint Fourier transform infrared (FTIR) spectra of (a) original extracts, (b) fermented extracts and (c) average hot water extract (red) and fermented extracts (blue). Plot (c) shows the major difference in the spectral region from 1200 to 950 cm−1 between the pre and post fermented extract. Figure reproduced in colour in online version. Further, the difference in the composition between HWE and AE is clear when the spectra of AE is subtracted from HWE (Fig. 2). With the production of alcohol in the AE samples, there is a region showing the presence of alcohol at 2976 cm−1. However, the most meaningful spectral changes are in the region between 1200 and 950 cm−1 , which is associated with the change in sugar profile as the yeast has consumed fermentable sugars, in particular maltose. Figure 2Open in figure viewerPowerPoint Difference in spectra between original extract and apparent extract samples. Major changes in absorbance in the region from 1200 to 950 cm−1 owing to utilization of sugars by the yeast during fermentation as well as the appearance of alcohol at 2976 cm−1 in the fermented worts. To further explore the changes between OE and AE samples, a PCA plot (Fig. 3a) shows the clear separation between the OE samples (blue) and AE samples (red) in PC1 (89%). The variation in G:L ratio within each mash style is represented in PC2 (11%). The arrow shows the direction from 1:2 to 1:4 G:L mashes. The larger difference is between the 1:2 G:L between the two mashes. This is further evidence showing the effects of high-temperature (infusion) vs. low-temperature ramping. Figure 3Open in figure viewerPowerPoint Principal component analysis (PCA) scores (a) and loadings plot (b) for original and fermented extracts. The arrow in Fig. 2(a) shows the G:L from 1:2 to 1:4. The major spectral region driving PC1 is shown in the loading plot (Fig. 3b) for the 1200–950 cm region, where ~1030 cm represents the influence (change) of maltose. The loading plot for PC2 (11%) represents changes in sugar concentration and differences between the individual sugars for differing G:L ratios (not shown). Table 5 shows the descriptive and calibration statistics for OE, AE and the three sugars. All calibrations had a strong coefficient of determination values of >0.98. As an example of the relationship of reference vs. predicted values of the calibrations, Fig. 4 shows the plot for maltose, indicating the good fit over the range of maltose values. The root mean square error of cross-validation values were very low for each and comparable to the standard error of the methods. These low values contributed to the high ratio of standard deviation to standard error (RPD) statistic. RPD values >5 indicate that the calibration would be suitable for quality control measures and could be applied in malthouse analysis or in-line measurement post mashing. Each calibration was built with five or six factors with very low bias. Table 5. Calibration statistics (all models developed using SNV 1d Savitsky-Golay) Parameter No of samples Mean SD SE R2 RMSECV RPD(cv) Factors Bias OE (°P) 24 10.94 3.2 0.25 0.9907 0.30 9.4 5 −3.2 × 107 AE (°P) 24 1.8 0.5 0.033 0.9952 0.04 12.5 5 −5.0 × 109 OE maltose (%) 24 5.79 1.6 0.12 0.9939 0.13 8.0 6 1.2 × 107 OE maltotriose (%) 24 1.22 0.6 0.025 0.9832 0.03 5.5 6 −3.4 × 109 OE glucose (%) 24 0.85 0.3 0.045 0.9803 0.05 15.1 5 3.2 × 108 SD, Standard deviation; SE, standard error; OE (°P), original extract as degrees Plato; AE (°P), apparent extract expressed as degrees Plato; R2, coefficient of determination; RMSECV, root mean standard error of cross-validation; RMSEP, root mean standard error of prediction; RPD, ratio RMSEP to standard deviation; factors, number of model components; Bias, deviation away from 1:1 line. Figure 4Open in figure viewerPowerPoint Original extract actual maltose levels (x-axis) vs. predicted maltose levels (y-axis) using all spectra. When building infrared calibrations, it important to understand the biochemical data within the sample set. The extract values and level of fermentable sugars measured in this study highlight the relationship between levels of extract and fermented extract, which allowed calibrations to be developed for extract and fermentable sugars. The two mashing experiments were designed to create a range of extracts and fermentable sugar levels that contributed to the positive calibration results. Of interest is the potential lack of a relationship between the level of AAL and the starting and final sugar profiles. While measuring AAL is usually an industry specification, perhaps there is additional value in knowing the levels of fermentable sugars, even if predicted by infrared spectroscopy. Acknowledgements Thanks go to Loraine Watson-Fox for assistance in mashing (experiment 1) and Dr. Jennifer Waanders for HPLC analysis of fermentable sugars. 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