Use of Response Surface Methodology to Investigate the Effectiveness of Commercial Enzymes on Buckwheat Malt for Brewing Purposes

Blaise P. Nic Phiarais 1,3, Beatus D. Schehl 1,3, Jorge C. Oliveira 2 and Elke K. Arendt 1,4
ABSTRACT

J. Inst. Brew. 112(4), 324332, 2006 The influence of two factors, total concentration and fraction of three pairs of commercial enzymes, which showed statistical significance (Biocellulase W with Hitempase 2XL, Biocellulase W with Amylo 300 and Amylo 300 with Hitempase 2XL), were studied for their overall effect on buckwheat wort quality using response surface methodology (RSM). This study revealed that the addition of increasing levels of Hitempase 2XL to the buckwheat mash increased colour, extract levels, wort filtration, fermentability and total fermentable extract (TFE), along with decreasing viscosity values. Results also determined a high level of fermentability when an enzyme combination of 30% Biocellulase and 70% Hitempase was added to the mash. The addition of increasing levels of Amylo 300 to buckwheat mashes resulted in increases in fermentability and total fermentable extract (TFE), along with increases in total soluble nitrogen (TSN), free amino nitrogen (FAN) and Kolbach index (KI). With regard to the proposed optimal regime, although no synergistic effect was found when the three enzymes were used together, the optimum conditions for the production of buckwheat wort with lowest viscosity, highest extract and optimal fermentability were achieved using a joint model. Overall, the findings of this study demonstrate the feasibility of producing wort suitable for the brewing of glutenfree beer from 100% malted buckwheat with careful optimisation of enzyme types and dosage levels. Key words: Buckwheat, enzyme activity, response surface methodology, wort quality.

INTRODUCTION
There is a renewed interest in buckwheat as an alternative crop for organic cultivation as well as an ingredient for health food products 22. Widely grown in Asia, Central
1 Department

of Food and Nutritional Sciences, National University of Ireland, University College Cork, College Road, Cork, Ireland. 2 Department of Process and Chemical Engineering, National University of Ireland, University College Cork, College Road, Cork, Ireland. 3 Biotransfer Unit, National University of Ireland, University College Cork, College Road, Cork, Ireland. 4 Corresponding author. E-mail: e.arendt@ucc.ie
Publication no. G-2006-1308-459 2006 The Institute of Brewing & Distilling

and Eastern Europe, Fagopyrum esculentum Moench (common buckwheat) is traditionally used for pasta, blended bread and other types of flour 11. Many positive physiological effects are associated with buckwheat. Due to the presence of soluble and insoluble fibres, antioxidants as well as the absence of glutenin-like proteins, buckwheat is considered a grain which can be used for preventative nutrition. Soluble and insoluble dietary fibres have a positive effect on constipation and obesity 12 whereas antioxidants may play an important role in lipid antioxidation and cancer prevention 20. Coeliac disease is an autoimmune enteropathy triggered by the ingestion of gluten-containing cereals in susceptible individuals 7. The worldwide incidence of coeliac disease is predicted to increase over the next years due to reinforced awareness of the disease and improved diagnostic and screening procedures 8. Treatment involves a gluten-free diet that avoids ingestion of cereals that contain gluten and their products 7. Therefore an increased demand for gluten-free products is expected. As it contains no glutenin-like proteins e.g. hordein, buckwheat is safe for patients who suffer from coeliac disease 5. Furthermore, buckwheat is becoming increasingly popular as a brewing ingredient. Maccagnan et al.14 recently used buckwheat, mainly as an unmalted adjunct in micro brewing, for the production of gluten-free beer. However, because of its low level of essential enzymes (e.g. -amylase, -amylase and -glucanases), mashing using 100% malted buckwheat (without the aid of commercial enzymes) can lead to problems, such as low extract yields, high wort viscosities, decreased rates of lautering and fermentation problems 17. To overcome these problems, MacFadden and Clayton 15 suggested various commercial enzyme additions. This involves the use of a range of proteases, -amylases, -amylases, -glucanases, amyloglucosidases, and cellulases. The purpose of this study was to determine the effects of increasing the dosages of the commercial enzymes Biocellulase W (a fungal cellulase), Amylo 300 (a fungal amyloglucosidase), Hitempase 2XL (a heat-stable -amylase), Bioxylanase 10L (a fungal xylanase), Bioferm L (a fungal -amylase), Bioglucanase B-10L (a bacterial glucanase) and Bioprotease N-100L (a bacterial protease) on wort quality when mashing with 100% malted buckwheat (Fagopyrum esculentum Moench). The goal was to

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identify the enzymic preparation that would result in an optimum brewing process. Response surface methodology (RSM) was used to investigate the effects of the total concentration and the fraction of each pair of three enzymes, Biocellulase W, Amylo 300 and Hitempase 2XL, on wort quality. The three enzymes were chosen based on preliminary results for the full set. The influence of total concentration and fraction of enzyme in a mixture on selected quality parameters was to be analyzed using a quadratic model to identify constrained and unconstrained points of optimum, which would then be related by subsequent experiments performed at the conditions predicted by the models to be optimum.

Mashing Malted buckwheat was mashed according to the optimised buckwheat mashing program as suggested by Wijngaard et al.24 All enzymes were added at mash-in unless otherwise stated. Wort analysis EBC-methods 3.3.2 and 4.3.2 were used to determine the total nitrogen (TN) level in flour of unmalted and malted samples, respectively 6. Total soluble nitrogen (TSN) determination was based on EBC-method 8.9.2 using a nitrogen analyzer (LECO type FP-528; LECO, St. Joseph, MI). Free amino nitrogen (FAN) in wort was measured according to EBC 8.10. The extract (%) of resulting wort was measured with a Servo-Chem Automatic Beer Analyzer (SCABA) obtained from Tecator AB, Sweden. Wort viscosity was measured using a falling ball viscometer (Haake, Karlsruhe, Germany) at 20C according to EBC-method 8.4. Colour was determined in accordance with EBC-method 8.5 and fermentability was based on EBC-method 8.6.1. The mash was filtered according to EBC-method 4.5.1, through filter paper (Schleicher & Schnell, Dassel, Germany) into graduated cylinders. The time taken for each wort sample to filter was recorded. Preliminary screening of enzymes Preliminary screening in a range of 0.051.5% concentration of enzymes was performed in triplicate to determine the optimum concentration range and effectiveness of the various enzymes, to select the most promising ones for subsequent study. From the data obtained, three enzymes were selected. Experimental procedure and data analysis of preliminary experiments All preliminary experiments were repeated three times. All samples were analyzed in duplicate. The results quoted are the mean of the three repeated experiments. Statistical data analysis (Students t-test, paired test) was performed using the SigmaPlot program (Systat Software UK Ltd, London, UK). P is the Probability and has a value from 0 to 1, where 00.25 is a significant difference. Experimental design and data analysis A response surface methodology (RSM) as described by Box et al.4 was applied to determine the relative contributions of two factors on the quality of buckwheat wort and beer. The two factors (process variables) considered were total enzyme concentration and fraction of enzyme in a binary mixture. It was decided to analyse separately

MATERIALS AND METHODS

Raw materials Unmalted common buckwheat (Fagopyrum esculentum) originating from an unspecified location in Eastern Europe and harvested in 2004 was obtained from Trouw B.V. (Rotterdam, The Netherlands). Malted buckwheat was found to have a moisture content of 6.1%, total nitrogen of 1.9% and friability of 91.7%. In all mashing experiments, distilled water was used. The commercial enzyme preparations Bioxylanase 10L, Bioferm L, Bioglucanase B-10L, Biocellulase W, Bioprotease N-100L, Amylo 300 and Hitempase 2XL were kindly provided by Kerry Biosciences Ltd. (Kilnagleary, Carrigaline, Co. Cork, Ireland). Details regarding the enzymatic activities of the enzyme preparations are outlined in Table I. Throughout this paper, the enzyme amounts are expressed as the dosage percentage (v/w) of grist. The calcium level of the mash-in liquor was adjusted to 200 ppm using calcium chloride (CaCl2 6H2O) and hydrochloric acid (HCL) was used to adjust the mash-in pH to 5.6, in order to increase the stability of the commercial enzymes. In-house research, yet to be published, determined the optimum calcium and pH levels. Malting procedure Malting was carried out using optimised procedures for steeping 26, germination 25 and kilning 17 using a micro malting machine (Joe White Malting Systems, Perth, Australia). Milling Malted buckwheat (Fagopyrum esculentum) was milled with a Bhler Miag laboratory-scale disc mill (Bhler GmbH, Braunschweig, Germany) set at a fine setting of 0.05 mm.
Table I. Commercial enzymes used in this study. Code A B C D E F G Commercial enzyme Biocellulase W Amylo 300 Hitempase 2XL Bioxylanase 10L Bioferm L Bioglucanase B-10L Bioprotease N-100L Derivative organism Trichoderma reesei Aspergillus niger Bacillus licheniformis Trichoderma reesei Aspergillus oryzae Bacillus subtilus Bacillus subtilus Description

pH optima 5.0 4.0 4.08.0 3.07.0 4.36.3 5.57.0 6.0

Temperature optima 60C 75C 90C 55C 5560C 60C 55C

Activity a 1,000 U /mL 1,100 U /mL 120,000 U /mL 10,000 U /mL 600,000 U /mL 10,000 U /mL 100,000 U /mL

Cellulase Amyloglucosidase -Amylase Xylanase -Amylase -Glucanase Protease

a Determined

by Kerry Bioscience (Kilnagleary, Carrigaline, Co. Cork, Ireland) standard methods.

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Table II. Circumscribed central composite experimental design used in this study. Coded Total concentration level 1 1 1 1 0 0 1/2 1/2 0 0 Value Total concentration (mL enzyme / 100 g grist) 0.1 0.1 1 1 0.55 0.55 0.232 0.868 0.55 0.55 Fraction of i in i + j mixture 0 1 0 1 0.15 0.85 0.5 0.5 0.5 0.5

Experiment no. 1 2 3 4 5 6 7 8 9 10

Level 1 1 1 1 1/2 1/2 0 0 0 0

Three pairs of enzymes were tested: i = A; j = B; i = A j = C; i = B j = C, where A is Biocellulase W, B is Amylo 300 and C is Hitempase 2XL. Experiments were performed in random order.

all mixtures of two enzymes rather than producing mixtures of 3 enzymes, to duly study the possible synergistic effects. A circumscribed central composite design was defined for each of the 3 pairs of the 3 enzymes selected. This experimental plan was defined by considering first the maximum and minimum values of the chosen variables on the basis of preliminary brewing tests, and these values were coded as 1 and 1, respectively. The arithmetic average between them defined the centre points of the solution space (coded value 0). The central composite experimental design requires two other settings of the variables, called star points, which for this variant of the design are located at the coded values 1/ 2. Table II shows the experimental design applied for each pair of enzymes. It is noted that experiments were performed in random order to minimise bias, and not in the order shown in the table. The responses (quality characteristics) measured for each case were extract, fermentability, viscosity, wort volume, colour, total soluble nitrogen (TSN), total fermentable extract (TFE) and Kolbach Index (KI). Each response for each enzyme pair was analysed by fitting quadratic models to the data, as indicated in equation 1, with least square regression.
R k,i, j = a k,i, j + b k,i, j Tc + c k,i, j Fc + d k,i, j Tc Fc + e k,i, j Tc2 + fk,i, j Fc2

(1) where Rk is the measured value for quality response k, Tc is the coded value of the total enzyme concentration, Fc the coded value of the fraction of enzyme i in the i + j mixture, and the model parameters a to f have the following meaning: a is the offset (average of all response values); b and c are half of the individual linear effects of factors T and F, respectively; e and f are half of the individual quadratic effects of factors T and F, respectively, and d half of the interactive effect of the two factors. As the model parameters have these meanings, regardless of the goodness of fit it is possible to analyse which of the 5 effects have statistical significance. Dividing each effect by the corresponding standard error of the model parameter gives the standardised effect, and those that are statistically significant are bigger than the t-Student value for 95% confidence level and 10 degrees of freedom (16 data points in each model 6 model parameters = 10). This
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information is neatly visualised in Pareto diagrams, but these are not shown for conciseness of the paper. The goodness of fit of equation 1 for each response and pair of enzymes was evaluated by the coefficient of determination R2, which is equal to the percentage of the variance of the data that is explained by the model. In RSM, values above 88% are usually demanded for the model to be usable for predictive purposes with minimum confidence, albeit limited to the solution space (extrapolation beyond the range of values tested experimentally is not recommended). In those cases, 3-D graphs can be plotted to visualise the solution space and help identify regions of optimum. Optimum values (settings of the two factors that give an absolute minimum or maximum value of a response) can be calculated analytically by finding the zeros of the derivatives of equation 1. For constrained optimisation (finding points of maximum or minimum of a response which obey specified limits of other responses), the Solver add-in of the Microsoft Excel software (XP version, Microsoft, Seattle, USA) was used, applying the Simplex algorithm of optimisation. For predictive purposes, a global model was also applied for each response, pooling all data together for the 3 pairs of enzymes, as indicated in equation 2. In this global model, the factors used were the actual concentration of each enzyme in v/w. It is noted that all enzyme concentrations are variables that range between 0 and 1, when expressed in mL enzyme /100 g of grist.
R k = k + A,k C A + B,k C B + C,k C C + AB,k C A C B + AC,k C A CC + BC,k C BC C + A,k C2 + B,k C2 + C,k C 2 A B C

(2)

where again the k subscript indicates the respective response, CA, CB and CC are the concentrations of enzymes A, B and C, respectively, in mL /100 g grist (the enzymes corresponding to each letter are given in Table I) and the model parameters (alpha) to (delta) are evidently related mathematically to the parameters of the 3 models for each pair. However, to avoid error propagation, the model parameters were obtained by least square regression of all data jointly for each response. This global model can be used for predictive purposes to identify which enzyme concentrations give an optimum result. It is

Table III. Preliminary enzyme trials showing the statistical significance of using enzymes according to the Students t-test, paired test. Enzyme A B C D E F G Extract (% dry wt) Positive 3 Positive 1 Positive 1 None None None Positive 2 Viscosity (mPas) None Positive 4 Positive 5 None None None Negative 1 Filtrate volume (ml) None Positive 4 Positive 5 None None None Negative 1 Fermentability (%) Positive 6 Positive 1 Positive 1 None None None Positive 2 TFE (g L1) Positive 3 Positive 1 Positive 1 None None None Positive 2

The enzymes (AF) are indicated in Table I. Enzyme concentrations where statistical significance was observed 1 0.05%1.5%, 2 1.5%, 3 1&1.5%, 4 0.05%, 5 0.51.5%, 6 0.11.5%, Filtrate volume after 30 min, TFE = Total Fermentable Extract.

Table IV. Statistical significance at a 95% confidence level of the effects of all enzyme pairs on all quality characteristics measured. Responses (quality characteristics) Pair A+B Effect T F (of A) TF T2 F2 R2 (%) T F (of A) TF T2 F2 R2 (%) T F (of B) TF T2 F2 R2 (%) Extract Fermentability yes yes yes 36.3 yes yes 97.3 yes yes yes 99.5 yes yes Viscosity yes yes yes 89.8 yes yes Wort volume yes yes Colour TSN FAN yes yes yes yes 34.9 yes yes yes 94.2 yes yes yes 94.9 32.1 98.8 yes 49.3 yes 80.3 yes yes 86.4 92.5 TFE yes yes yes yes 99.7 yes yes yes yes 99.4 yes yes yes 97.9 85.1 31.6 KI

91.5

A+C

93.0

89.9

74.4

48.7 yes

B+C

76.5

93.4

67.6

65.7

Enzymes are denoted A, B and C as per Table I. T indicates the linear effect of total enzyme concentration, F the linear effect of the fraction of enzyme i in the i + j mixture (I = A or B, j = B or C), T F indicates the interactive effect and T2 and F2 the quadratic effects. The coefficients of determination (R2) are also shown, bold meaning sufficiently high value for the model to be acceptable for predictive purposes.

noted that the model was built from experimental data with pairs of enzymes only and therefore it is only applicable to ternary mixtures if the three-level interactive effect is negligible. In RSM, this would be a reasonable assumption, as it is a third order term.

et al.10, the decrease in filtration of the sample is related to the degrading effect that Bioprotease N-100L has on malt amylases thus resulting in a retarded efficient liquefaction. Effect of the enzymes on quality characteristics The effects that data analysis indicated to be statistically significant are shown in Table IV. This table gives a condensed view of the full set of data, as it would be excessive to show the Pareto diagrams for all responses and pairs of enzymes. It is noted that when the model provides a poor fit, the standard errors of the parameters are very large and therefore effects tend to be statistically negligible. However, the true meaning of this situation is that the system is influenced by other factors more, or as significantly as the two chosen, and it does not necessarily imply that the factors have no effect. The responses that generally had better model fits were fermentability, FAN and TFE. Viscosity was well fitted for the pairs containing enzyme A, while colour was well fitted for the two pairs containing enzyme C. Interactive effects were often significant. Inspection of the nature of these effects showed that they were due to a synergistic effect between the two enzymes being significant only at
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RESULTS AND DISCUSSION

Preliminary results It is clear from Table III that enzyme additions of Bioxylanase 10L, Bioferm L and Bioglucanase B-10L did not accelerate wort filtration or improve extract development, wort viscosity and fermentability significantly when compared to the control. Bajoma and Young 1 using sorghum malt (20%) and unmalted sorghum (80%) with the use of these enzymes, reported similar results. Commercial enzymes that gave significantly improved extract, fermentability and TFE, when compared to the control to which no enzymes were added, were selected for further trials. These included Biocellulase W, Amylo 300 and Hitempase 2XL. Although Bioprotease N-100L showed significantly improved extract, fermentability and TFE, the rate of filtration was adversely affected. According to Goode

higher total concentrations. One quadratic effect on TFE was significant for all pairs, which may indicate that a point of optimum exists within the range of concentrations tested. Figures 1 to 3 show the 3-D surfaces for all cases where the surface fit is acceptable (R2 in bold in Table IV), enabling a better visualisation. Extract It can be seen from Fig. 1a, that there was an increase in extract associated with increasing the fraction and concentration of Hitempase 2XL, when virtually no Biocellulase W was present. This supports the findings of Goode et al.10, where the extract increased significantly as the Hitempase 2XL levels of unmalted sorghum mashes were increased. The interactive effect of Biocellulase and Amylo 300, and Amylo 300 and Hitempase showed no significant effect on extract (data not shown). Viscosity and filtration performance A high viscosity makes beer filtration more difficult and may lead to starch hazes in the final beer 13. It is as a result of this that low viscosity wort is a desirable attribute of worts. Fig. 1b depicts a decrease in viscosity as the concentration of Hitempase 2XL is increased. The lowest viscosity was observed when 100% of Hitempase 2XL was used. Also noted was a slightly higher wort filtration (data not shown). These results are in agreement with previous studies using Hitempase 2XL carried out on unmalted sorghum 10. It was also noted that the lowest viscosity was associated with lowest concentration of Amylo 300, when no Biocellulase W was present (Fig. 2a). This supports the findings of Bajomo and Young 1, who found

that the lowest viscosity was evident when the lowest concentration of Amylo 300 was added to a mash containing 100% raw sorghum. An accompanying increase in wort volume was observed (Fig. 2b). This is in keeping with Siebert et al.21, who reported that viscosity impacted on wort filtration. The interactive effect of Amylo 300 and Hitempase showed no significant effect on viscosity and wort filtration (data not shown). Colour It is evident that with an increase in the fraction and concentration of Hitempase 2XL, there was an increase in the colour values of the subsequent worts (Figs. 1c and 3a). This increase in colour value of the worts was presumably because of the increased extraction of solubles in the wort. This concurs with evidence from Goode et al.10 who also found that increasing the levels of Hitempase 2XL in the raw sorghum mashes, resulted in increased wort colour values. The interactive effect of Biocellulase W and Amylo 300 imparted no effect on wort colour (data not shown). Nitrogenous fractions (TSN, KI, FAN) Yeast growth involves the uptake of nitrogen, mainly in the form of amino acids, for the synthesis cellular proteins and other cell components 18. Raw sorghum wort and barley malt wort should not contain a FAN level less than 100150 mg/L to ensure a healthy fermentation 3. Previous work has shown that when buckwheat malt was optimally mashed, similar levels were found, i.e. a FAN level of ~125 mg/L 16.

Fig. 1. Effects of the total concentration of Biocellulase W and Hitempase 2XL and the fraction of Biocellulase W in a Biocellulase W and Hitempase 2XL mixture, where 1.0 is 100% of Biocellulase W on (a) extract (R2 = 93%), (b) viscosity (R2 = 89.8%), (c) colour (R2 = 94.2%), (d) fermentablity (R2 = 99.5%) and (e) total fermentable extract (TFE) (R2 = 99.5%).
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Fig. 2. Effects of the total concentration of Biocellulase W and Amylo 300 and the fraction of Biocellulase W in a Biocellulase W and Amylo 300 mixture, where 1.0 is 100% of Biocellulase W on (a) viscosity (R2 = 89.9%), (b) wort volume (R2 = 91.5%), (c) free amino nitrogen (FAN) (R2 = 98.8%), (d) fermentability (R2 = 97.3%) and (e) total fermentable extract (TFE) (R2 = 99.7%).

Fig. 3. Effects of the total concentration of Amylo 300 and Hitempase 2XL and the fraction of Amylo 300 in a Amylo 300 and Hitempase 2XL mixture, where 1.0 is 100% of Amylo 300 on (a) colour (R2 = 95%) (b) total soluble nitrogen (TSN) (R2 = 86.4%), (c) kolbach index (KI) (R2 = 95.1%), (d) free amino nitrogen (FAN) (R2 = 92.5%), (e) fermentability (R2 = 93.4%), and (f) total fermentable extract (TFE) (R2 = 97.7%).
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Results indicate that the highest levels in FAN were obtained from the maximum concentration of Amylo 300, with no other enzyme present (Fig. 2c). However, this does not correlate with previous work carried out with raw sorghum and Amylo 300, where Amylo 300 imparted little or no effect on the levels of FAN in the wort 3. No significant effect was noted from the interactive effect of Biocellulase W and Amylo 300 with regard to TSN (data not shown). However, it was found that the addition of Hitempase 2XL had a negative effect on TSN at high concentrations (Fig. 3b). This concurs with previous studies, using Hitempase 2XL, carried out on unmalted sorghum 10. It was also noted that TSN levels were positively influenced by the addition of high levels of Amylo 300. This is further confirmed by values obtained for the Kolbach index (3c), which is the ratio of soluble protein to total protein, thus providing a reasonable index of the extent of protein degradation 9. Results obtained for FAN levels reflect the same trend (Fig. 3d). This supports the idea that as more protein was solubilised, this protein was degraded to FAN (amino acids and peptides) during mashing. Fermentability and total fermentable extract (TFE) Previous studies indicate that the low levels of fermentable sugars obtained from buckwheat malt were as a result of low amylolytic activity generated during germination. The quality of malt and wort is considerably compromised when malt contains low amylolytic levels 16,17. The highest level of fermentability was obtained using approximately 30% Biocellulase W in combination with 70% Hitempase 2XL (Fig. 1d). In addition, the curves appeared to approach limiting values. This implies that further additions of the enzymes would have had little effect on fermentability. Results obtained for extract and fermentability levels reflect the same trend observed in total fermentable extract (Fig. 1e). It has been observed by many workers 2,3,19,23 that an increase in the total concentration of Amylo 300 results in a corresponding increase in fermentability. The results presented in Fig 2d support these findings. Amylo 300 degrades dextrins into simpler fermentable carbohydrate moieties and complements intrinsic -amylase acivity 19.

Using Amylo 300 alone in the mash at an increased total concentration gave the highest fermentability. Correlating total fermentable extracts can be seen in Fig. 2e. It was found that when Hitempase 2XL was combined with Amylo 300, it did not contribute significantly to fermentability (Fig. 3e), but did yield higher levels of fermentable extract (Fig. 3f) in mid range concentration. However at all times Amylo 300 when used alone produced the highest levels of fermentability and total fermentable extract. Prediction and validation of optimum conditions Pooling all data from the 3 pairs of enzymes in a single regression using equation 2 for each response provided the results shown in Table V. Parameters that are statistically significant at a 95% confidence level are indicated in bold typeface (i.e. whose value divided by the standard error is greater than the t-Student value for 95% confidence level and 14 degrees of freedom: 24 data points minus 10 model parameters). It can be seen that this model predicts that Biocellulase W (enzyme A) does not influence the results significantly compared to the other enzymes, and its only effects of relevance are an interaction with Hitemase 2XL (enzyme C), when mixed with it, affecting colour, and with Amylo 300, affecting the extract. The concentration of both Amylo 300 and Hitempase 2XL affect the quality characteristics that are better fitted by the surface model: fermentability, colour, FAN and TFE, and quadratic effects are relevant in all cases, which suggests that points of optimum may exist. It can be seen that model predictions can be made with acceptable confidence for fermentability, colour, FAN and TFE. A number of optimum search scenarios were defined. The concentrations of enzymes that according to the models provide the best solution for each scenario are shown in Table VI. It is noted that the predictions for viscosity and extract objectives are made using models that have a low certainty (R2 below 88%, although above 73%), but given their importance, they were also considered. Further experiments were then performed using the enzyme concentrations thus specified, with some replicates, and the results are also shown in Table VI. It can be seen that the model predictions were indeed very good, even in the

Table V. Parameters of the predictive global surface model relating quality characteristics to the concentration of the three selected enzymes. Responses (quality characteristics) Coefficient off-set CA CB CC CA CB CA CC CB C C CA2 CB2 CC2 R2 (%) Extract 71.6586 6.6333 8.0529 12.0391 32.4592 16.6995 16.5666 5.1077 5.8852 7.3490 73.0 Fermentability 69.0174 2.2595 40.6605 31.2610 14.0727 2.0951 32.9141 2.8493 21.7692 23.7362 93.3 Viscosity 2.4778 0.1150 0.01480 0.07178 0.4353 0.7628 0.4167 0.08970 0.008428 0.1157 78.6 Wort volume 285.9216 44.6750 20.1980 26.2906 65.3797 184.4113 22.0091 16.2263 15.6305 45.2455 54.0 Colour 4.5061 0.003292 0.1298 0.8941 0.2944 0.8496 0.01218 0.03389 0.1098 0.4592 95.9 TSN 825.186 45.730 287.874 86.621 418.845 309.800 75.628 17.063 208.694 155.427 66.0 FAN 144.269 6.0106 44.5372 18.1170 21.9977 21.9977 15.7091 0.8256 24.4835 30.2383 95.4 TFE 49.3835 3.4408 36.0893 31.8790 16.3314 11.2197 36.1991 6.0488 20.5603 22.7902 93.3 KI 29.3056 1.9164 10.5965 3.6361 16.3086 11.7905 3.3986 0.3864 7.6786 5.8916 65.2

Parameters that are statistically significant are indicated in bold and shaded background. The coefficients of determination (R2) are also shown, bold meaning sufficiently high value for the model to be acceptable for predictive purposes.

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Table VI. Enzyme concentrations predicted by the models to fulfill the specified objective and results of the validation experiments. Enzyme conc. (ml enzyme / 100 g grist) Objective Lowest viscosity1 Highest TFE2 Highest extract with fermentability constraint3 Highest extract with fermentability constraint3 Highest TFE1 Highest TFE1 Highest TFE with fermentability constraint1 Highest TFE with fermentability constraint1
1 as

Extract Model prediction 76.6 76.6 Experimental result 76.5 76.9

Fermentability Model prediction 84.0 84.0 84.0 84.0 Experimental result 83.2 83.0 85.9 85.7 Model prediction 64.9 65.3 65.3 64.0 64.0

TFE Experimental result 66.1 65.7 65.6 65.0 64.5

Viscosity Model prediction 2.22 Experimental result 2.25

A 0.36 0 0 0 0 0 0 0

B 0.64 0.98 0.49 0.49 0.86 0.86 0.62 0.62

C 0 0.02 0 0 0 0 0 0

predicted by the joint model, 2 as predicted by the B + C model, 3 as predicted by the A + C model.

cases where the model R2 was lower. It can also be seen that the predictions of the global model (equation 2 with the parameters of Table V) were actually the best, and therefore this model is recommended.

CONCLUSIONS
In this study, the effects of 7 individual enzymes (Biocellulase W, Amylo 300, Hitempase 2XL, Bioxylanase 10L, Bioferm L, Bioglucanase B-10L and Bioprotease N100L) on the quality of wort when mashing with 100% malted buckwheat (Fagopyrum esculentum Moench) were investigated. Further to this, the influence of two factors, total concentration and fraction of three pairs of enzymes, which showed statistical significance (Biocellulase W with Hitempase 2XL, Biocellulase W with Amylo 300 and Amylo 300 with Hitempase 2XL), were studied for their overall effect on the buckwheat wort quality using response surface methodology. This study revealed that the addition of increasing levels of Hitempase 2XL to the buckwheat mash increased colour, extract levels, wort filtration, fermentability and TFE, along with decreasing viscosity values. Fermentability and TFE appeared to approach limiting values which was expected. The addition of Amylo 300 increased >20% the amount of fermentable carbohydrate in sorghum malt, which is due to the degradation of dextrins into smaller fermentable carbohydrate moieties and complements intrinsic -amylase activity by an amyglucosidase enzyme 2,19. As expected, the addition of increasing levels of Amylo 300 to buckwheat mashes resulted in corresponding increases in fermentability and TFE, along with increases in TSN, FAN and KI. Results also determined a high level of fermentability when 30% of Biocellulase and 70% of Hitempase was added to the mash. In practice, the most important objective would be to achieve the highest TFE with fermentability in the range of 8284% 6. The response surface model predicted that this is achieved using Amylo 300 alone, in a concentration of 0.62 mL /100 g of grist, and this was validated experi-

mentally. The model predicts that fermentability is at the highest value of the range defined (84%), although the experimental results gave a higher figure. Therefore, a safety margin should be considered, lowering the concentration to 0.55 mL /100 g of grist. The viscosity of this wort is rather high. The mean value stated by EBC for wort viscosity is 1.867 mPas 6. It can be decreased somewhat by adding 0.36 mL /100 g grist of Biocellulase W, although this reduces the TFE and still results in a higher viscosity than desired. This work shows the usefulness of a simple experimental design and response surface methodology in identifying optimum conditions for brewing with a limited amount of experimental data. It is noted that the method is based on deterministic functions, that is, the effect of process variability is not considered. Other methods and more work would be required to minimise product variability.
ACKNOWLEDGEMENTS

This project was funded by the Irish Government under the National Development Plan, 20062007. The authors gratefully acknowledge Eoin Lalor (Kerry Bioscience Ireland Ltd.) for kindly providing the commercial enzyme preparations and Dr. Helge Ulmer for his assistance with the experimental design and statistical analysis.
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