- Email: [email protected]
Characterization of fermentation of waste wheat-rye bread mashes with the addition of complex enzymatic preparations
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
Available online at www.sciencedirect.com
http://www.elsevier.com/locate/biombioe
Characterization of fermentation of waste wheat-rye bread mashes with the addition of complex enzymatic preparations Joanna Kawa-Rygielska a,*, Witold Pietrzak a, Anna Czubaszek b skiego 37/41, Department of Food Storage and Technology, Wrocław University of Enviromental and Life Sciences, Chełmon Wrocław, Poland b Department of Fruit, Vegetable and Cereals Technology, Wrocław University of Enviromental and Life Sciences, Poland a
article info
abstract
Article history:
There were prepared mashes from wheat-rye bread residues at concentration of 32% (w/w).
Received 19 March 2011
In a control sample amylolytic enzymes were used (a-amylase and glucoamylase). Studied
Received in revised form
samples were prepared using complex enzymatic preparations Cremix 2XL and Ceremix 6X
24 January 2012
MG containing proteases and enzymes degrading non-starch polysaccharides. Mashes
Accepted 16 April 2012
were inoculated with Saccharomyces cerevisiae Ethanol Red yeast and subjected to the
Available online 14 May 2012
fermentation. The addition of complex preparations led to shortening the fermentation time to 72 h in comparison to control sample (93 h). Initial glucose concentration was
Keywords:
similar in all samples, while the addition of Ceremix preparations increased the concen-
Ethanol fuel
tration of maltose and maltotriose in obtained mashes. In fermentation media prepared
Fermentation
with supportive enzymes carbohydrates were consumed by yeast at a higher rate. Highest
Wheat-rye bread residues
glycerol concentration was formed in fermentation feed prepared with Ceremix 6X MG
Starch
(8.01 gL 1). Acetic acid concentration was similar in all samples during the fermentation
Hydrolysis
(ca. 0.5 gL 1), while most of succinic acid was formed in the control media (2.11 gL
Mashing
69 h of process). The most advantageous physiological condition of yeast biomass after
1
after
fermentation process featured the media prepared with Ceremix 6X MG (75.35% of budding cells, 5,88% inactive cells). The addition of Ceremix 2XL and Ceremix 6X MG preparations resulted in significant increase in ethanol yield in wheat-rye bread mashes (35.53 and 36,60 g ethanol per 100 g of bread dry matter respectively) in comparison to control sample (35.24 g ethanol per 100 g of bread dry matter). ª 2012 Published by Elsevier Ltd.
1.
Introduction
Due to continuously increasing prices of crops suitable as raw material for ethanol production (corn, cereals, potatoes) it is necessary to search for new, inexpensive fermentation feed. Food industry wastes seem to be one of the most suitable materials to be used in distilleries, and among them bakery waste deserve special attention. As previously mentioned waste wheat bread can be used to produce ethanol [1]. In Poland most common bakery product is wheat-rye bread.
Its annual production is almost 170 thousand tons, of which approximately 10% will be returned to the bakery. In the European Union, percentage of wastes produced by the bakery industry is reported to be above 7% of its total production [2] what may be approximately 1,3 million thons annually. Some returns are being processed for the purpose of bread crumbs production, or as animal feed, while significant amounts are not suitable for reprocessing due to mold contamination, significant level of staling or other reasons. There are also studies on the use of waste bread as raw
* Corresponding author. E-mail address: [email protected] (J. Kawa-Rygielska). 0961-9534/$ e see front matter ª 2012 Published by Elsevier Ltd. doi:10.1016/j.biombioe.2012.04.016
18
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
material for the purpose of: lactic acid production [3], biohydrogen production by anaerobic fermentation [4], production of aroma compounds by yeast [5] and others. During the process of bread baking, starch is subjected to significant changes. Part of it is gelatinized and partly depolymerized, which may facilitate its subsequent hydrolysis during mashing. Although part of bread starch is bound in combination with other ingredients of flour, which makes its extraction and distribution complicated [6]. A solution to this problem may be additional enzymatic hydrolysis by complex preparations containing, apart from amylases, enzymes that break down nonstarch polysaccharides and proteins. It may cause an increase in available carbohydrates for the yeast and hydrolysis of gluten network which may release the bound starch and provide free amino acids, which are the source of nitrogen for yeast [7]. The aim of present study was to inspect the ethanol yield and the fermentation course of residual wheat-rye bread mashes by the addition of complex enzymatic preparations before liquefaction and saccharification stage. Carbohydrate profiles of bread mashes obtained using traditional method and with the addition of supportive enzymes were compared. Suitability of manufactured substrates was determined by the assessment of dynamics of the fermentation process, the rate and extent of carbohydrate consumption by yeast and the amount of metabolites formed.
Suspension of partially fragmented raw material (80 g bread, 150 mL distilled water) Adjusting the pH to 6.0 (30% NaOH)
1
Materials and methods
2.1.
Raw materials
Wheat-rye bread (after shelf live, returns from shops) was obtained from local bakery. Raw material showed no signs of mold infestation. Bread was partially fragmented into cubes of about 1 cm. The moisture of material was measured using WPS 50P weighing dryer (Radwag, Poland) and was 26.26%. Starch content in investigated raw material was measured due to polarymetric technique according to BS EN ISO 10520:1998 [8]. Its average content ranged 51.17%.
2.2.
Enzymes and yeast
Liquefaction enzyme (Termamyl SC) containing thermostable aamylase was provided by Novozymes, Denmark. Saccharification enzymes Spiryzyme Fuel (glucoamylase) and Optimash BG (hemicellulase) were provided by Genencor, USA. Complex enzymatic preparations Ceremix 2XL (a-amylase, b-glucanase and protease) and Ceremix 6X MG (a-amylase, b-glucanase, protease, pentosanase and cellulase) used in the preliminary phase were provided by Novozymes. Commercial distillery dried yeast strain Saccharomyces cerevisiae Ethanol Red was obtained from Fermentis, France. Yeast strain used in present study contained 38,07% budding cells, none inactive cells were found.
2.3.
2
Liquefaction T=85°C, t=60 min Termamyl SC (0,4 mL)
0- control sample 1- Ceremix 6X MG (0,1 g) 2- Ceremix 2XL (0,3 mL)
Saccharification T=55°C, t=90 min, pH=5,8 50 % H2SO4 Spiryzyme Fuel (0,96 mL) Optimash BG (0,4 mL)
Cooling T=20°C Supplementing with distilled water, pH correction (50% H2SO4) m=250 g, pH=5,0
Fig. 1 e Diagram of mashing process.
2.4.
2.
Preliminary phase T=45°C, t=20 min
0
Inoculation and fermentation
Mashes were inoculated with 2 gL 1 S. cerevisiae Ethanol Red dry mass and fermented in 300 mL Erlenmeyer flasks. Yeast were rehydrated prior to inoculation in 100 mL of sterile water for 30 min. The fermentation was performed using periodic method at 30 C. Samples were thermostated until no signs of CO2 emission were shown. Fermentation tests were performed in triplicate.
2.5.
Analytical methods
The fermentation dynamics and the time of fermentation was determined on the basis of mass changes during the fermentation process (until differences in mass measurements were less than 0.05 g). Physiological condition of yeast cells was determined microscopically in Thoma chamber after staining with methylene blue. Carbohydrate profiles of mashes and fermenting liquids (glucose, maltose and maltotriose), fermentation by-products (glycerol, succinic and acetic acid) and ethanol concentration were determined by HPLC using D-7000 HPLC chromatograph (Merck, Germany) with ion exchange column HPX-87 H (Bio-Rad, USA) at 60 C with refractive index detector. The mobile phase was 0.005 M H2SO4 at a flow rate of 0.6 mL min 1. Ethanol yield from 100 g starch (Y(g1 et. 100 g starch)) and from 100 g of initial bread dry mass (Y(g1et. 100 g dry mass)) were calculated.
Mashing process
Preparation of mashes from waste wheat-rye bread was made using laboratory mashing device LB Electronic (Lochner Labor, Germany). The experiments were carried out with mash concentration of 32% (w/w). A detailed diagram of mashing process is shown in Fig. 1.
3.
Results
3.1.
Fermentation dynamics
Fermentation dynamics, assesed by the amount of released CO2, is one of the primary determinants of yeast fermentation
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
activity in investigated media. Amount of carbon dioxide released after particular hours of fermentation, described as percentage of total CO2 released, is shown in Fig. 2. Fermentation of control sample ended after 93 h. Usage of additional enzymatic preparations during mashing process led to significant reduction of fermentation time. Fermentation of samples additionally hydrolyzed with Ceremix 2XL and Ceremix 6X MG lasted 72 h.
3.2.
Carbohydrate profiles during fermentation
During the fermentation yeast consumed almost all available sugars in fermentation media (Fig. 3). The initial glucose concentration ranged between 122.41 gL 1 (Ceremix 6X MG) and 132.79 gL 1 (Ceremix 2XL). During the first day of the process yeast consumed glucose at a similar rate. In control sample concentration of glucose after 45 h of process was 76.13 gL 1, yet in samples additionally treated with Ceremix 2XL the amount of glucose was lower (49.29 gL 1). After the same time only traces of glucose were left in the sample mashed with Ceremix 6X MG. When process of fermentation was completed, less than 0.5 gL 1 glucose remained in all samples. After mashing process maltose concentration was highest in samples mashed with Ceremix 6X MG addition (11.15 gL 1). During the first 21 h of fermentation maltose content increased in all samples and ranged from 14.99 gL 1 to 10.79 gL 1. On the second day of fermentation in the control sample the quantity of maltose slightly decreased. The concentration of maltose in samples additionally treated with Ceremix enzyme preparations was reducing after the first day of the process to final amount of about 1.6 gL 1. After completion of the fermentation, maltose content in the control sample was 1.01 gL 1. The concentration of maltotriose after mashing process was highest in control sample (1.08 gL 1). In the samples additionally treated with Ceremix 2XL and 6X MG maltotriose level was considerably lower (0,38 and 0.56 gL 1 respectively). After 21 h of fermentation maltotriose level was similar in all samples (ca. 0.8 gL 1). During next days of fermentation the concentration of maltotriose was constantly decreasing. The final concentration of maltotriose were between 0.14 gL 1 (Control) and 0.05 gL 1 (Ceremix 6X MG).
100 Control Ceremix 2XL
CO2 [% of total]
80
Ceremix 6X MG
60
40
20
0 21
45
72
93
Time [h] Fig. 2 e Dynamics of CO2 release during wheat-rye bread media at concentarion of 32% (w/w).
19
3.3. Effect of additional enzymatic treatment on ethanol formation dynamics The addition of complex enzymatic preparations improved ethanol formation dynamics (Fig. 4.). After the first day of fermentation the highest amount of ethanol formed in media prepared with addition of Ceremix 6X MG (40.98 gL 1). In the control media the concentration of ethanol after the first day ranged 21.22 gL 1. During the second day of fermentation the concentration of ethanol increased at a similar rate in media mashed with the addition of Ceremix preparations. The rate of ethanol formation decreased after 45 h of fermentation in samples prepared with Ceremix 2XL and 6X MG. In control sample the rate of ethanol formation decreased after 70 h of process.
3.4.
Final effects of fermentation
Table 1 shows the final effects results of fermentation of waste wheat-rye bread mashes. Final concentration of ethanol in control sample, mashed only with standard liquefaction and saccharification enzymes, was 83.10 gL 1. The addition of Ceremix 2XL and 6X MG preparations in mashing process allowed to increase the amount of ethanol produced to 85,80 and 88.50 gL 1 respectively. In the control sample the yield of ethanol ranged 35,24 g per 100 g dry matter of raw material. The introduction of Ceremix 2XL allowed to increase yield to 35,53 g ethanol per 100 g of raw material. The best results of ethanol yield were obtained for the sample mashed with Ceremix 6X MG preparation. Process efficiency for this sample increased to 36,60 g ethanol per 100 g of bread dry matter.
3.5. Effect of additional enzymatic treatment on the formation of by-products during fermentation During fermentation of wheat-rye bread mashes typical fermentation by-products (glycerol, organic acids) were formed. After the first day of the process the lowest amount of glycerol (3.69 gL 1) was found in the control sample (Fig. 5A). In the sample additionally hydrolyzed with Ceremix 2XL the concentration of glycerol was slightly higher (4.50 gL 1). The highest amount of glycerol after 21 h of fermentation (7.01 gL 1) was formed in the sample made with Ceremix 6X MG. During subsequent days of the process, the concentration of glycerol in control sample and the sample mashed with the addition of Ceremix 2XL was similar. Glycerol concentration in the sample previously mashed with Ceremix 6X MG addition exceeded the level of 8.0 gL 1, and not virtually did change till the end of the process. There were formed small amounts of acetic acid during fermentation of mashes obtained from wheat-rye bread (Fig. 5B). Its concentration ranged between 0.4e0.6 gL 1, and did not significantly change during fermentation. After the first day of fermentation the highest amount of succinic acid was formed in the sample additionally mashed with Ceremix 6X MG (1.03 gL 1). In the second day of the process the concentration of succinic acid greatly increased in control sample (from 0.67 to 1.89 gL 1). Amount of succinic acid in the sample obtained with Ceremix 2XL did not change in comparison to initial state. The concentration of succinic acid raised in all samples at the end of fermentation.
20
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
A
140 Ceremix 2XL
-1
Glucose [gL ]
Control
120 100
Ceremix 6X MG
80 60 40 20 0 0
B
10
20
30
40
50
60
70
80
100
16 Control Ceremix 2XL Ceremix 6X MG
Maltose [gL ]
14 -1
90
12 10 8 6 4 2 0 0
20
30
40
50
60
70
80
90
100
1,2 Control
1
-1
Maltotriose [gL ]
C
10
Ceremix 2XL
0,8
Ceremix 6X MG
0,6 0,4 0,2 0 0
10
20
30
40
50
60
70
80
90
100
Time [h] Fig. 3 e Effect of additional hydrolysis by Ceremix 2XL and Ceremix 6X MG on carbohydrates (glucose (A), maltose (B), maltotriose (C)) consumption in fermenting mashes made from waste wheat-rye bread.
3.6. Effect of the modification of mashing process on physiological condition of yeast cells during fermentation S. cerevisiae Ethanol Red yeast strain, used in this study, was characterized by a good physiological condition after fermentation process (Fig. 6). At the end of the fermentation process a number of budding cells in the control sample did not change significantly. In the samples with Ceremix preparations the content 90
4.
Discussion
It has been known that the use of additional technological treatments can increase the efficiency of ethanol fermentation process of traditional materials such as corn or potatoes [9e12].
75
Ethanol [gL -1]
of budding cells greatly increased in comparison to the initial state. After completing the fermentation process some inactive cells were found in fermentation media. Their content was between ca. 5.70% (control, Ceremix 6X MG) and 12.70% (Ceremix 2XL).
60
Table 1 e Ethanol concentration and final process yield of fermentation of whet-rye bread mashes at concentration of 32% (w/w).
45
30
Ethanol [gL 1]
Control
15
Ceremix 2XL Ceremix 6X MG
Ethanol yield Y(g
et.
100 g starch
1 )
Y(g et. 100 g dry mass
0 0
10
20
30
40
50
60
70
80
90
100
Time [h]
Fig. 4 e Dynamics of ethanol formation in wheat-rye bread mashes at concentration of 32% (w/w) prepared with the addition of Ceremix 2XL and Ceremix 6X MG.
Control Ceremix 2XL Ceremix 6X MG
83.10 85.80
50.76 52.41
35.24 35.53
88.50
54.06
36.60
1 )
21
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
A
9 8
-1
Glycerol [gL ]
7 6 5 4 3 Control
2
Ceremix 2XL
1
Ceremix 6X MG
0 0
10
20
30
40
50
60
70
80
90
100
Time [h]
B
2,5
-1
Concentration [gL ]
Control 2
Ceremix 2XL Ceremix 6X MG
1,5
1
0,5
0
21h
45h
69h
93h
ac etic ac id
21h
45h
69h
93h
s uc c inic ac id
Fig. 5 e Effect of Ceremix 2XL and Ceremix 6X MG addition during mashing process on formation of glycerol (A) and acetic and succinic acid (B) in fermenting wheat-rye bread mashes.
Although there are few works dealing with the use of food waste to produce ethanol [1,13,14]. The use of waste as raw material in the distillery requires knowledge of their chemical composition and transformation they are subjected to during processing. In the course of bread production and storage, its ingredients are subjected to significant changes including formation of lipid-gluten network [15], Maillard reactions and starch retrogradation [16,17]. These changes could reduce suitability 80
Number of cells [% of total]
70
budding cells inactive cells
60 50 40 30 20 10 0 Control
Ceremix 2XL
Ceremix 6X MG
Fig. 6 e Physiological condition of Saccharomyces cerevisiae Ethanol Red yeast strain after fermentation of mashes prepared from waste wheat-rye bread at a concentration of 32% (w/w).
of bread as fermentation feed. As has been previously mentioned, wheat bread is an efficient feedstock for ethanol production [1]. Present study has shown that wheat-rye bread could also become high yielding raw material for fermentation. Moreover, the use of supportive enzyme preparations Ceremix 2XL and Ceremix 6X MG led to further increase of process efficiency. The use of preparations containing active protease could cause disruption of the gluten network of bread which could make more bound starch available for enzymatic hydrolysis. This fact confirms higher maltose and maltotriose content in the mashes prepared with Ceremix preparations. Moreover, hydrolysis of proteins available in used raw material could increase the amount of amino acids in fermentation media, which is essential for proper physiological condition and activity of yeast. Rye flour contains about 9% of pentosans which also form crosslinked structures with other ingredients of bread [18]. Ceremix 6X MG preparation, containing pentosanase, could also affect its presence and make more carbohydrates and proteins soluble. According to Kłosowski et al. [9], the addition of enzyme preparations, containing proteases and enzymes degrading non-starch polysaccharides, markedly increases fermentation of corn mashes productivity. Application of these preparations during the mashing of food waste materials may be necessary to achieve high productivity due to alterations in raw material during processing. On the basis of the results obtained, bakery waste can be used as a raw material in distilleries. However, it should be
22
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
noted that vast majority of waste products are not segregated and may not constitute a homogeneous material. Furthermore, the problem is the proper storage of raw material. As previously mentioned contagion of raw material by mold reduce its technological value [19]. This problem concerns in particular waste materials such as bread. However impact of these factors on the suitability of waste bread for ethanol production requires further study.
references
[1] Ebrahimi F, Khanahmadi M, Roodpeyma S, Taherzadeh MJ. Ethanol production from bread residues. Biomass Bioenerg 2008;32:333e7. [2] Mena C, Adenso-Diaz B, Yurt O. The causes of food waste in the supplier-retailer interface: evidences from the UK and Spain. Resour Conserv Recy 2011;55:648e58. [3] Oda Y, Park BS, Moon KH, Tonomura K. Recycling of bakery wastes using an amylolytic lactic acid bacterium. Bioresour Technol 1997;60:101e6. [4] Doi T, Matsumoto H, Abe J, Morita S. Feasibility study on the application of rhizosphere microflora of rice for the biohydrogen production from wasted bread. Int J Hydrogen Energ 2009;34:1735e43. [5] Daigle P, Gelinas P, Leblanc D, Morin A. Production of aroma compounds by Geotrichum candidum on waste bread crumbs. Food Microbiol 1999;16:517e22. [6] McCann T, Small DM, Batey IL, Wrigley CW, Day L. Proteinlipid interactions in gluten elucidated acetic acid fractionation. Food Chem 2009;115:105e12. [7] Yue G, Ju J, Zhang X, Tan T. The influence of nitrogen source on ethanol production by yeast from concentrated sweet sorghum juice. Biomass Bioenerg 2012;39:48e52. [8] BS EN ISO 10520:1998. Native starch. Determination of starch content. Ewers polarimetric method, ISBN: 0 580 30395 0.
ski B, Kotarska K. [9] Kłosowski G, Mikulski D, Czupryn Characterisation of fermentation of high-gravity maize mashes with the application of pullulanase, proteolytic enzymes and enzymes degrading non-starch polysaccharides. J Biosci Bioeng 2010;5:466e71. [10] Srichuwong S, Fujiwara M, Wang X, Seyama T, Shiroma R, Arakane M, et al. Simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash for the production of ethanol. Biomass Bioenerg 2009;33: 890e8. [11] Karimi K, Emtiazi G, Taherzadeh MJ. Ethanol production from dilute-acid pretreated rice straw by simultaneous saccharification and fermentation with Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. Enzyme Microb Tech 2006;40:138e44. [12] Devantier R, Pedersen S, Ollson L. Characterization of very high gravity ethanol fermentation of corn mash. Effect of glucoamylase dosage, pre-saccharification and yeast strain. Appl Microbiol Biot 2005;68:622e9. [13] Prasad S, Singh A, Joshi HC. Ethanol as an alternative fuel from agricultural, industrial and urban residues. Resour Conservation Recycling 2007;50:1e39. [14] Salhofer S, Obersteiner G, Schneider F, Lebersorger S. Potentials for the prevention of municipal solid waste. Waste Manage 2008;28:245e59. [15] Singh H. A study of changes in wheat protein during bread baking using SE-HPLC. Food Chem 2005;1-2:247e50. [16] Haralampu SG. Resistant starch- a review of the physical properties and biological impact of RS3. Carbohyd Polym 2000;41:285e92. [17] Ribotta PD, Le Bail A. Thermo-physical assessment of bread during staling. LWT Food Sci Technol 2007;40:879e84. [18] Buksa K, Nowotna A, Praznik W, Gambu s H, Ziobro R, Krawontka J. The role of pentosans and starch in baking wholemeal rye bread. Food Res Int 2010;43:2045e51. [19] Kawa-Rygielska J, Chmielewska J, Pla˛skowska E. Effect of raw material quality on fermentation activity of distillery yeast. Pol J Food Nutr Sci 2007;57:275e9.
Available online at www.sciencedirect.com
http://www.elsevier.com/locate/biombioe
Characterization of fermentation of waste wheat-rye bread mashes with the addition of complex enzymatic preparations Joanna Kawa-Rygielska a,*, Witold Pietrzak a, Anna Czubaszek b skiego 37/41, Department of Food Storage and Technology, Wrocław University of Enviromental and Life Sciences, Chełmon Wrocław, Poland b Department of Fruit, Vegetable and Cereals Technology, Wrocław University of Enviromental and Life Sciences, Poland a
article info
abstract
Article history:
There were prepared mashes from wheat-rye bread residues at concentration of 32% (w/w).
Received 19 March 2011
In a control sample amylolytic enzymes were used (a-amylase and glucoamylase). Studied
Received in revised form
samples were prepared using complex enzymatic preparations Cremix 2XL and Ceremix 6X
24 January 2012
MG containing proteases and enzymes degrading non-starch polysaccharides. Mashes
Accepted 16 April 2012
were inoculated with Saccharomyces cerevisiae Ethanol Red yeast and subjected to the
Available online 14 May 2012
fermentation. The addition of complex preparations led to shortening the fermentation time to 72 h in comparison to control sample (93 h). Initial glucose concentration was
Keywords:
similar in all samples, while the addition of Ceremix preparations increased the concen-
Ethanol fuel
tration of maltose and maltotriose in obtained mashes. In fermentation media prepared
Fermentation
with supportive enzymes carbohydrates were consumed by yeast at a higher rate. Highest
Wheat-rye bread residues
glycerol concentration was formed in fermentation feed prepared with Ceremix 6X MG
Starch
(8.01 gL 1). Acetic acid concentration was similar in all samples during the fermentation
Hydrolysis
(ca. 0.5 gL 1), while most of succinic acid was formed in the control media (2.11 gL
Mashing
69 h of process). The most advantageous physiological condition of yeast biomass after
1
after
fermentation process featured the media prepared with Ceremix 6X MG (75.35% of budding cells, 5,88% inactive cells). The addition of Ceremix 2XL and Ceremix 6X MG preparations resulted in significant increase in ethanol yield in wheat-rye bread mashes (35.53 and 36,60 g ethanol per 100 g of bread dry matter respectively) in comparison to control sample (35.24 g ethanol per 100 g of bread dry matter). ª 2012 Published by Elsevier Ltd.
1.
Introduction
Due to continuously increasing prices of crops suitable as raw material for ethanol production (corn, cereals, potatoes) it is necessary to search for new, inexpensive fermentation feed. Food industry wastes seem to be one of the most suitable materials to be used in distilleries, and among them bakery waste deserve special attention. As previously mentioned waste wheat bread can be used to produce ethanol [1]. In Poland most common bakery product is wheat-rye bread.
Its annual production is almost 170 thousand tons, of which approximately 10% will be returned to the bakery. In the European Union, percentage of wastes produced by the bakery industry is reported to be above 7% of its total production [2] what may be approximately 1,3 million thons annually. Some returns are being processed for the purpose of bread crumbs production, or as animal feed, while significant amounts are not suitable for reprocessing due to mold contamination, significant level of staling or other reasons. There are also studies on the use of waste bread as raw
* Corresponding author. E-mail address: [email protected] (J. Kawa-Rygielska). 0961-9534/$ e see front matter ª 2012 Published by Elsevier Ltd. doi:10.1016/j.biombioe.2012.04.016
18
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
material for the purpose of: lactic acid production [3], biohydrogen production by anaerobic fermentation [4], production of aroma compounds by yeast [5] and others. During the process of bread baking, starch is subjected to significant changes. Part of it is gelatinized and partly depolymerized, which may facilitate its subsequent hydrolysis during mashing. Although part of bread starch is bound in combination with other ingredients of flour, which makes its extraction and distribution complicated [6]. A solution to this problem may be additional enzymatic hydrolysis by complex preparations containing, apart from amylases, enzymes that break down nonstarch polysaccharides and proteins. It may cause an increase in available carbohydrates for the yeast and hydrolysis of gluten network which may release the bound starch and provide free amino acids, which are the source of nitrogen for yeast [7]. The aim of present study was to inspect the ethanol yield and the fermentation course of residual wheat-rye bread mashes by the addition of complex enzymatic preparations before liquefaction and saccharification stage. Carbohydrate profiles of bread mashes obtained using traditional method and with the addition of supportive enzymes were compared. Suitability of manufactured substrates was determined by the assessment of dynamics of the fermentation process, the rate and extent of carbohydrate consumption by yeast and the amount of metabolites formed.
Suspension of partially fragmented raw material (80 g bread, 150 mL distilled water) Adjusting the pH to 6.0 (30% NaOH)
1
Materials and methods
2.1.
Raw materials
Wheat-rye bread (after shelf live, returns from shops) was obtained from local bakery. Raw material showed no signs of mold infestation. Bread was partially fragmented into cubes of about 1 cm. The moisture of material was measured using WPS 50P weighing dryer (Radwag, Poland) and was 26.26%. Starch content in investigated raw material was measured due to polarymetric technique according to BS EN ISO 10520:1998 [8]. Its average content ranged 51.17%.
2.2.
Enzymes and yeast
Liquefaction enzyme (Termamyl SC) containing thermostable aamylase was provided by Novozymes, Denmark. Saccharification enzymes Spiryzyme Fuel (glucoamylase) and Optimash BG (hemicellulase) were provided by Genencor, USA. Complex enzymatic preparations Ceremix 2XL (a-amylase, b-glucanase and protease) and Ceremix 6X MG (a-amylase, b-glucanase, protease, pentosanase and cellulase) used in the preliminary phase were provided by Novozymes. Commercial distillery dried yeast strain Saccharomyces cerevisiae Ethanol Red was obtained from Fermentis, France. Yeast strain used in present study contained 38,07% budding cells, none inactive cells were found.
2.3.
2
Liquefaction T=85°C, t=60 min Termamyl SC (0,4 mL)
0- control sample 1- Ceremix 6X MG (0,1 g) 2- Ceremix 2XL (0,3 mL)
Saccharification T=55°C, t=90 min, pH=5,8 50 % H2SO4 Spiryzyme Fuel (0,96 mL) Optimash BG (0,4 mL)
Cooling T=20°C Supplementing with distilled water, pH correction (50% H2SO4) m=250 g, pH=5,0
Fig. 1 e Diagram of mashing process.
2.4.
2.
Preliminary phase T=45°C, t=20 min
0
Inoculation and fermentation
Mashes were inoculated with 2 gL 1 S. cerevisiae Ethanol Red dry mass and fermented in 300 mL Erlenmeyer flasks. Yeast were rehydrated prior to inoculation in 100 mL of sterile water for 30 min. The fermentation was performed using periodic method at 30 C. Samples were thermostated until no signs of CO2 emission were shown. Fermentation tests were performed in triplicate.
2.5.
Analytical methods
The fermentation dynamics and the time of fermentation was determined on the basis of mass changes during the fermentation process (until differences in mass measurements were less than 0.05 g). Physiological condition of yeast cells was determined microscopically in Thoma chamber after staining with methylene blue. Carbohydrate profiles of mashes and fermenting liquids (glucose, maltose and maltotriose), fermentation by-products (glycerol, succinic and acetic acid) and ethanol concentration were determined by HPLC using D-7000 HPLC chromatograph (Merck, Germany) with ion exchange column HPX-87 H (Bio-Rad, USA) at 60 C with refractive index detector. The mobile phase was 0.005 M H2SO4 at a flow rate of 0.6 mL min 1. Ethanol yield from 100 g starch (Y(g1 et. 100 g starch)) and from 100 g of initial bread dry mass (Y(g1et. 100 g dry mass)) were calculated.
Mashing process
Preparation of mashes from waste wheat-rye bread was made using laboratory mashing device LB Electronic (Lochner Labor, Germany). The experiments were carried out with mash concentration of 32% (w/w). A detailed diagram of mashing process is shown in Fig. 1.
3.
Results
3.1.
Fermentation dynamics
Fermentation dynamics, assesed by the amount of released CO2, is one of the primary determinants of yeast fermentation
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
activity in investigated media. Amount of carbon dioxide released after particular hours of fermentation, described as percentage of total CO2 released, is shown in Fig. 2. Fermentation of control sample ended after 93 h. Usage of additional enzymatic preparations during mashing process led to significant reduction of fermentation time. Fermentation of samples additionally hydrolyzed with Ceremix 2XL and Ceremix 6X MG lasted 72 h.
3.2.
Carbohydrate profiles during fermentation
During the fermentation yeast consumed almost all available sugars in fermentation media (Fig. 3). The initial glucose concentration ranged between 122.41 gL 1 (Ceremix 6X MG) and 132.79 gL 1 (Ceremix 2XL). During the first day of the process yeast consumed glucose at a similar rate. In control sample concentration of glucose after 45 h of process was 76.13 gL 1, yet in samples additionally treated with Ceremix 2XL the amount of glucose was lower (49.29 gL 1). After the same time only traces of glucose were left in the sample mashed with Ceremix 6X MG. When process of fermentation was completed, less than 0.5 gL 1 glucose remained in all samples. After mashing process maltose concentration was highest in samples mashed with Ceremix 6X MG addition (11.15 gL 1). During the first 21 h of fermentation maltose content increased in all samples and ranged from 14.99 gL 1 to 10.79 gL 1. On the second day of fermentation in the control sample the quantity of maltose slightly decreased. The concentration of maltose in samples additionally treated with Ceremix enzyme preparations was reducing after the first day of the process to final amount of about 1.6 gL 1. After completion of the fermentation, maltose content in the control sample was 1.01 gL 1. The concentration of maltotriose after mashing process was highest in control sample (1.08 gL 1). In the samples additionally treated with Ceremix 2XL and 6X MG maltotriose level was considerably lower (0,38 and 0.56 gL 1 respectively). After 21 h of fermentation maltotriose level was similar in all samples (ca. 0.8 gL 1). During next days of fermentation the concentration of maltotriose was constantly decreasing. The final concentration of maltotriose were between 0.14 gL 1 (Control) and 0.05 gL 1 (Ceremix 6X MG).
100 Control Ceremix 2XL
CO2 [% of total]
80
Ceremix 6X MG
60
40
20
0 21
45
72
93
Time [h] Fig. 2 e Dynamics of CO2 release during wheat-rye bread media at concentarion of 32% (w/w).
19
3.3. Effect of additional enzymatic treatment on ethanol formation dynamics The addition of complex enzymatic preparations improved ethanol formation dynamics (Fig. 4.). After the first day of fermentation the highest amount of ethanol formed in media prepared with addition of Ceremix 6X MG (40.98 gL 1). In the control media the concentration of ethanol after the first day ranged 21.22 gL 1. During the second day of fermentation the concentration of ethanol increased at a similar rate in media mashed with the addition of Ceremix preparations. The rate of ethanol formation decreased after 45 h of fermentation in samples prepared with Ceremix 2XL and 6X MG. In control sample the rate of ethanol formation decreased after 70 h of process.
3.4.
Final effects of fermentation
Table 1 shows the final effects results of fermentation of waste wheat-rye bread mashes. Final concentration of ethanol in control sample, mashed only with standard liquefaction and saccharification enzymes, was 83.10 gL 1. The addition of Ceremix 2XL and 6X MG preparations in mashing process allowed to increase the amount of ethanol produced to 85,80 and 88.50 gL 1 respectively. In the control sample the yield of ethanol ranged 35,24 g per 100 g dry matter of raw material. The introduction of Ceremix 2XL allowed to increase yield to 35,53 g ethanol per 100 g of raw material. The best results of ethanol yield were obtained for the sample mashed with Ceremix 6X MG preparation. Process efficiency for this sample increased to 36,60 g ethanol per 100 g of bread dry matter.
3.5. Effect of additional enzymatic treatment on the formation of by-products during fermentation During fermentation of wheat-rye bread mashes typical fermentation by-products (glycerol, organic acids) were formed. After the first day of the process the lowest amount of glycerol (3.69 gL 1) was found in the control sample (Fig. 5A). In the sample additionally hydrolyzed with Ceremix 2XL the concentration of glycerol was slightly higher (4.50 gL 1). The highest amount of glycerol after 21 h of fermentation (7.01 gL 1) was formed in the sample made with Ceremix 6X MG. During subsequent days of the process, the concentration of glycerol in control sample and the sample mashed with the addition of Ceremix 2XL was similar. Glycerol concentration in the sample previously mashed with Ceremix 6X MG addition exceeded the level of 8.0 gL 1, and not virtually did change till the end of the process. There were formed small amounts of acetic acid during fermentation of mashes obtained from wheat-rye bread (Fig. 5B). Its concentration ranged between 0.4e0.6 gL 1, and did not significantly change during fermentation. After the first day of fermentation the highest amount of succinic acid was formed in the sample additionally mashed with Ceremix 6X MG (1.03 gL 1). In the second day of the process the concentration of succinic acid greatly increased in control sample (from 0.67 to 1.89 gL 1). Amount of succinic acid in the sample obtained with Ceremix 2XL did not change in comparison to initial state. The concentration of succinic acid raised in all samples at the end of fermentation.
20
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
A
140 Ceremix 2XL
-1
Glucose [gL ]
Control
120 100
Ceremix 6X MG
80 60 40 20 0 0
B
10
20
30
40
50
60
70
80
100
16 Control Ceremix 2XL Ceremix 6X MG
Maltose [gL ]
14 -1
90
12 10 8 6 4 2 0 0
20
30
40
50
60
70
80
90
100
1,2 Control
1
-1
Maltotriose [gL ]
C
10
Ceremix 2XL
0,8
Ceremix 6X MG
0,6 0,4 0,2 0 0
10
20
30
40
50
60
70
80
90
100
Time [h] Fig. 3 e Effect of additional hydrolysis by Ceremix 2XL and Ceremix 6X MG on carbohydrates (glucose (A), maltose (B), maltotriose (C)) consumption in fermenting mashes made from waste wheat-rye bread.
3.6. Effect of the modification of mashing process on physiological condition of yeast cells during fermentation S. cerevisiae Ethanol Red yeast strain, used in this study, was characterized by a good physiological condition after fermentation process (Fig. 6). At the end of the fermentation process a number of budding cells in the control sample did not change significantly. In the samples with Ceremix preparations the content 90
4.
Discussion
It has been known that the use of additional technological treatments can increase the efficiency of ethanol fermentation process of traditional materials such as corn or potatoes [9e12].
75
Ethanol [gL -1]
of budding cells greatly increased in comparison to the initial state. After completing the fermentation process some inactive cells were found in fermentation media. Their content was between ca. 5.70% (control, Ceremix 6X MG) and 12.70% (Ceremix 2XL).
60
Table 1 e Ethanol concentration and final process yield of fermentation of whet-rye bread mashes at concentration of 32% (w/w).
45
30
Ethanol [gL 1]
Control
15
Ceremix 2XL Ceremix 6X MG
Ethanol yield Y(g
et.
100 g starch
1 )
Y(g et. 100 g dry mass
0 0
10
20
30
40
50
60
70
80
90
100
Time [h]
Fig. 4 e Dynamics of ethanol formation in wheat-rye bread mashes at concentration of 32% (w/w) prepared with the addition of Ceremix 2XL and Ceremix 6X MG.
Control Ceremix 2XL Ceremix 6X MG
83.10 85.80
50.76 52.41
35.24 35.53
88.50
54.06
36.60
1 )
21
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
A
9 8
-1
Glycerol [gL ]
7 6 5 4 3 Control
2
Ceremix 2XL
1
Ceremix 6X MG
0 0
10
20
30
40
50
60
70
80
90
100
Time [h]
B
2,5
-1
Concentration [gL ]
Control 2
Ceremix 2XL Ceremix 6X MG
1,5
1
0,5
0
21h
45h
69h
93h
ac etic ac id
21h
45h
69h
93h
s uc c inic ac id
Fig. 5 e Effect of Ceremix 2XL and Ceremix 6X MG addition during mashing process on formation of glycerol (A) and acetic and succinic acid (B) in fermenting wheat-rye bread mashes.
Although there are few works dealing with the use of food waste to produce ethanol [1,13,14]. The use of waste as raw material in the distillery requires knowledge of their chemical composition and transformation they are subjected to during processing. In the course of bread production and storage, its ingredients are subjected to significant changes including formation of lipid-gluten network [15], Maillard reactions and starch retrogradation [16,17]. These changes could reduce suitability 80
Number of cells [% of total]
70
budding cells inactive cells
60 50 40 30 20 10 0 Control
Ceremix 2XL
Ceremix 6X MG
Fig. 6 e Physiological condition of Saccharomyces cerevisiae Ethanol Red yeast strain after fermentation of mashes prepared from waste wheat-rye bread at a concentration of 32% (w/w).
of bread as fermentation feed. As has been previously mentioned, wheat bread is an efficient feedstock for ethanol production [1]. Present study has shown that wheat-rye bread could also become high yielding raw material for fermentation. Moreover, the use of supportive enzyme preparations Ceremix 2XL and Ceremix 6X MG led to further increase of process efficiency. The use of preparations containing active protease could cause disruption of the gluten network of bread which could make more bound starch available for enzymatic hydrolysis. This fact confirms higher maltose and maltotriose content in the mashes prepared with Ceremix preparations. Moreover, hydrolysis of proteins available in used raw material could increase the amount of amino acids in fermentation media, which is essential for proper physiological condition and activity of yeast. Rye flour contains about 9% of pentosans which also form crosslinked structures with other ingredients of bread [18]. Ceremix 6X MG preparation, containing pentosanase, could also affect its presence and make more carbohydrates and proteins soluble. According to Kłosowski et al. [9], the addition of enzyme preparations, containing proteases and enzymes degrading non-starch polysaccharides, markedly increases fermentation of corn mashes productivity. Application of these preparations during the mashing of food waste materials may be necessary to achieve high productivity due to alterations in raw material during processing. On the basis of the results obtained, bakery waste can be used as a raw material in distilleries. However, it should be
22
b i o m a s s a n d b i o e n e r g y 4 4 ( 2 0 1 2 ) 1 7 e2 2
noted that vast majority of waste products are not segregated and may not constitute a homogeneous material. Furthermore, the problem is the proper storage of raw material. As previously mentioned contagion of raw material by mold reduce its technological value [19]. This problem concerns in particular waste materials such as bread. However impact of these factors on the suitability of waste bread for ethanol production requires further study.
references
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