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Profiling brewers' spent grain for composition and microbial ecology at the site of production
LWT - Food Science and Technology 43 (2010) 890–896
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Profiling brewers’ spent grain for composition and microbial ecology at the site of production James A. Robertson a, *, Kerry J.A. I’Anson b, Janneke Treimo c, Craig B. Faulds a, Tim F. Brocklehurst b, Vincent G.H. Eijsink c, Keith W. Waldron a a b c
Sustainable Food Chain Exploitation Platform, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom Microbial Ecology Exploitation Platform, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, N-1432 Aas, Norway
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2009 Received in revised form 14 January 2010 Accepted 19 January 2010
Brewers’ spent grain (BSG) is a readily available, high volume low cost byproduct of brewing and is a potentially valuable resource for industrial exploitation. The variation in BSG composition and the implications for microbiological spoilage by a resident microflora might affect the potential to use BSG as a reliable food-grade industrial feedstock for value-added downstream processing. Fresh samples of BSG from a range of 10 breweries have been analysed for their microbial and chemical composition. The results show that a resident microflora of mainly thermophilic aerobic bacteria (<107 g-1 fresh weight) persists on BSG. This population is susceptible to rapid change but at the point of production BSG can be considered microbiologically stable. Chemically, BSG is rich in polysaccharides, protein and lignin. Residual starch can contribute up to 13% of the dry weight and BSG from lager malts has higher protein content than that from ale. In general, at the point of production, BSG is a relatively uniform chemical feedstock available for industrial upgrading. Differences between breweries should not present problems when considering BSG for industrial exploitation but susceptibility to microbial colonisation is identified as a potential problem area which might restrict its successful exploitation. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Keywords: Brewers’spent grain Barley Microbiology Polysaccharide Phenolic Protein
1. Introduction Brewers’ spent grain (BSG) is obtained from barley (Hordeum vulgare L.) as the outer pericarp-seed coat layers from the original malted barley grain which remain after hot water extraction at 65– 70 C (mashing) (Mussatto, Dragone, & Roberto, 2006). It can represent approximately 30% (w/w) of the starting malted grain (Townsley, 1979), which makes BSG a readily available, high volume and low cost byproduct within the brewing industry. Although difficulties persist in the extraction of component polysaccharides and proteins (Celus, Brijs, & Delcour, 2006; Forssell et al., 2008; Treimo et al., 2009) BSG remains a potentially more valuable resource for industrial exploitation than the current general use as an animal feed. Indeed, value-added products are increasingly being sought from BSG but information on microbiological safety and its uniformity for use as an industrial feedstock can limit its appeal for exploitation.
* Corresponding author. Tel.: þ44 1603 255000; fax: þ44 1603 507723. E-mail address: [email protected] (J.A. Robertson).
Whilst the gross composition of BSG samples has been well documented (Mussatto et al., 2006; Santos, Jime´nez, Bartolome´, Go´mez-Cordove´s, & del Nozal, 2003; Valverde, 1994) less is known about the expected variation in the composition of BSG between breweries and the extent of its microbial contamination on production. Certainly the rich polysaccharide and protein content and the high moisture content of BSG makes it particularly susceptible to microbial growth and subsequent spoilage (Valverde, 1994). Where storage may be required for downstream processing of BSG then deterioration through microbial activity is perceived as a potential problem, unless the BSG can be stabilised post-production. Variation in BSG composition can be expected to arise from differences in barley malting cultivars, malting practices, adjuncts added and wort production during mashing processes in breweries. A major difference is that different barley cultivars are used as the malt source for lager and ale. Also, in general ale malt is kilned at a higher temperature (Palmer, 2006) whilst lager malts are derived from barley with a higher protein content (Briggs, 1998). Spent grain is often mixed with yeast, removed post-fermentation, and cold break protein (trub) separated from wort. Samples provided by breweries were obtained directly from the mash tun and hence the effects of yeast or trub was not considered.
0023-6438/$ – see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.01.019
J.A. Robertson et al. / LWT - Food Science and Technology 43 (2010) 890–896
In the current study the composition and resident microflora of BSG samples, obtained on the day of production, from a range of 10 breweries have been screened to estimate variation between breweries. This complements a previous study, where estimation of variation was limited to a within brewery comparison on composition (Santos et al., 2003). The results from the current study indicate to what extent BSG can be considered a reliable and uniform industrial feedstock for value-added downstream processing. 2. Materials and methods 2.1. Materials BSG from a range of 10 commercial breweries throughout the EU and South Africa was obtained as a single bulk sample from each brewery. Samples were the residues resulting from wort prepared from malted barley, for either lager or ale production. The BSG was supplied fresh from the mash tun, chilled to w4 C to facilitate microbiological analysis, unless otherwise stated. Samples of the BSG supplied were also freeze dried and milled to pass a 0.5 mm sieve prior to use for experimental analysis. All chemicals used were at least Analar quality. 2.2. Methods 2.2.1. Microbiology The naturally-associated microflora was enumerated using methods described by the International Commission on the Microbiological Specifications for Foods (ICMSF, 1978). In summary, within 24 h of production and delivery to the laboratory, samples of BSG (40 g fresh weight) were blended with peptone salt dilution fluid (PSDF) (360 mL) in a Stomacher Lab-blender 400 (Seward Laboratory) for 2 min. Viable micro-organisms from each sample were enumerated by plating serial dilutions of each supernatant, made in PSDF, onto the surface of a range of culture media using a Spiral Plate Maker. The culture media, incubation conditions, and the presumptive identification of micro-organisms enumerated on each medium were as shown in Table 1. Where further refinement of the presumptive identification was required, bacteria were subjected to standard Gram’s stain, and oxidase and catalase tests. 2.2.2. Moisture determination Samples of fresh BSG were dried to constant weight (16 h) at 104 C in a forced draught oven to determine dry weight/g fresh weight. 2.2.3. Alcohol-insoluble residues (AIR) preparation AIR were prepared prior to analysis for cell wall sugars, lignin, cell wall bound phenolic acids, starch and mixed-linkage b-glucans. Wet BSG (100 g; w22% dry weight; stored up to 24 h at 4 C) was Table 1 Presumptive groups of microflora enumerated from BSG. Presumptive Group
Plating medium
Aerobic mesophilic Plate Count Agar (PCA) bacteria Aerobic thermophilic Plate Count Agar (PCA) bacteria Pseudomonas spp. Cephaloridine, Fucidin, Cetrimide Agar (CFC) Yeasts and moulds Oxytetracycline, Dextrose, Yeast Extract Agar (ODY) Microaerophilic De Man, Rogosa, Sharpe bacteria Agar (MRS) Strictly anaerobic Oxygen-free Reinforced bacteria Clostridial Medium (RCM)
Incubation conditions In air at 20 C In air at 55 C In air at 25 C In air at 20 C Gaseous atmosphere of 90% H2 and 10% CO2 at 25 C Gaseous atmosphere of 90% H2 and 10% CO2 at 25 C
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homogenised for 30 s then plunged into boiling absolute ethanol (300 mL: w80% ethanol final concentration) for 1 h. The insoluble residue was recovered by filtration (GF/C paper using a Buchner funnel). After a further 2 extractions in boiling 70% ethanol (v/v) (each 300 mL; 2 h) the residue was extracted (2) in boiling absolute ethanol (300 mL; 5 min), then washed with cold absolute ethanol (150 mL). An aliquot of the final filtrate was tested for residual non-cell wall soluble sugars using phenol-sulphuric acid (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), to give a final filtrate containing less than 10 mg mL1 sugar. The insoluble residue (AIR) was washed in 2 volumes acetone (2), with washings removed by gentle suction, prior to drying to constant weight at 40 C. Except for protein analysis and starch maltodextrins all chemical analyses were performed on AIRs. 2.2.4. Sugars determination Sugars were determined as total sugars released from the sample AIR through Saeman hydrolysis, (pre-hydrolysis using 12 M H2SO4: 3 h room temperature followed by dilution to 1 M H2SO4) and hydrolysis for 1 h at 105 C). Hydrolysates were derivatised as their alditol acetates, and analysed by GLC using a flame ionisation detector (Blakeney, Harris, Henry, & Stone, 1983). Total uronic acid content was determined photometrically using glucuronic acid as a standard (Blumenkranz & Asboe-Hansen, 1973). 2.2.5. Klason lignin determination Lignin was determined as Klason lignin. Samples (w50 mg) were dispersed in 0.75 mL of 12 M sulphuric acid and incubated at room temperature (3 h) prior to dilution to 1 M acid concentration. After a further incubation (100 C for 2.5 h in sealed tubes) the insoluble residue was recovered by filtration (No. 3 glass sinter) under gentle suction and washed free of acid. Klason lignin was determined as the weight gain in each glass sinter after drying at 50 C to constant weight in a temperature controlled oven. 2.2.6. Cell wall bound phenolic acids determination Total alkali-extractable phenolic acid present in the samples were determined following saponification with 4 M NaOH (w5 mg mL1; 18 h; room temperature in the dark). Supernatants were recovered by centrifugation. An aliquot (0.8 mL) was acidified (pH w 2) using concentrated HCl, prior to extraction with ethyl acetate (3 3 mL). Extracts were reduced to dryness under nitrogen, resuspended in methanol: H2O (50/50, v/v) and constituent alkali soluble phenolic acids were separated and quantified using HPLC (LUNA C18 reverse phase HPLC column (Phenonomex, Macclesfield, UK)) according to the method of Waldron, Parr, Ng, and Ralph (1996), using trans-cinnamic acid as internal standard. 2.2.7. Starch determination Starch was determined as total starch (K-TSTA: Megazyme International Ireland Ltd.) based on the method of McCleary, Gibson, & Mugford, 1997. In brief, starch was measured as glucose following sample dispersion in DMSO, digestion using a thermostable a-amylase, followed by amyloglucosidase treatment and photometric quantification of glucose released. The assay conditions for starch assumed a starch content <100 mg g1 BSG in a10 mL reaction volume. Freeze dried samples were used to determine total starch, as starch þ dextrins, and AIR to distinguish polymeric starch. Amylose was determined using Megazyme kit AM/AMP 01/96. Samples were dispersed in DMSO and starch recovered through ethanol precipitation. After redissolution, amylopectin was specifically removed from solution using Con A and the supernatant was analysed for starch (amylose) through digestion with amylase/amyloglucosidase and quantification of glucose released.
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J.A. Robertson et al. / LWT - Food Science and Technology 43 (2010) 890–896
Table 2 Microbiological characterization of brewers’ spent grain samples. Log10 micro-organisms/g fresh weight brewers’ spent grain
Media/Micro-organisms
Sample code
PCA 20 C Aerobic Mesophilic bacteria PCA 55 C Aerobic Thermophilic bacteria CFC 25 C Pseudomonas spp. bacteria ODY 20 C Yeasts & moulds bacteria MRS 25 C Microaerophilic bacteria RCM 25 C Anaerobic bacteria
P01 004
P01 003
P02 001
P05 001
P08 001
P09 001
P11 001
2.58 3.06 <2 <2 <2 <2
2.90–5.33 5.33–6.60 <2 2.66–3.18 <2 2.43–4.76
3.59–4.43 6.00–7.00 <2 3.45–4.56 7.84–7.93 5.58–6.14
3.00–4.70 2.25–4.00 <2 <2 <2 1.95–3.00
3.10–3.60 2.00–2.30 <2 <2 <2 <2
5.90–6.12 3.96–5.30 <2 1.96–2.20 >7 >7
2.60–3.20 2.60–3.20 <2 <2 2.40–2.50 2.50–3.30
Sample code ¼ unique identifier used for sample traceability to specific brewery
2.2.8. Mixed-linkage b-glucan determination Mixed-linkage b-glucan, [(1-3),(1-4)-b-D-glucan], was assayed through selective degradation of the (1-3) linkage using lichenase (a specific endo-(1-3),(1-4)-b-D-glucan 4-glucanohydrolase) followed by release of glucose, using b-glucosidase, and its photometric assay using glucose oxidase, (K-BGLU: Megazyme International Ireland Ltd.). The kit procedure, based on 1 g sample weight was modified for use with a 100 mg sample weight. 2.2.9. Total nitrogen determination Nitrogen content of BSG was quantified using the Kjeldahl method. The samples were prepared and measured on a Kjeltec Auto 1035 sampler system (Tecator, Nerliens, Oslo, Norway) as described by Adler-Nissen (1986). Protein content was calculated from nitrogen using a conversion factor of 6.25 (Jones, 1941). 2.2.10. Amino acid composition analysis The amino acid composition of freeze dried BSG analyses was determined by the Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences according to standard EU methods (EC, Commission Directive 98/64/EC, 1998; EC, Commission Directive 2000/45/EC, 2000). In brief, a sample quantity corresponding to 10 mg nitrogen (N) was treated with a performic acid/phenol mixture to oxidise S-containing amino acids, hydrolysed with 6 M HCl for 23 h, pH-adjusted to 2.2, diluted with 0.2 M sodium citrate loading buffer, pH 2.2 (Biochrom Ltd., Cambridge, UK) and micro-filtrated (0.45 mm Spartan membrane filter, Schleicher & Schuell, Dassel, Germany) prior to analysis on a Biochrom 30 Amino Acid Analyser (Biochrom Ltd.) equipped with an Oxidized Feedstuff High Performance PEEK Column (Biochrom Ltd.). Amino acids were detected and quantified photometrically at 440 and 570 nm after post-column
derivatisation with ninhydrin. Data were analysed against appropriate external standards (Sigma Chemical, St. Louis, Mo., U.S.A.) using the ChromeleonÒ Chromatography Management Software (Dionex Corporation, Sunnyvale, CA). Tryptophan was analysed by HPLC (Summit HPLC System; Dionex Corp.), equipped with a SUPELCOSILÔ LC-18 column (Supelco, Bellefonte, Pa, USA) and a fluorescence detector (Shimadzu RF – 535, Shimadzu Corporation, Kyoto, Japan), after alkaline hydrolysis (EC, Commission Directive 2000/45/EC, 2000). Data were analysed against appropriate external standards (DL-Tryptophan and alphamethyl-tryptophan; Sigma Chemical) using the ChromeleonÒ Chromatography Management Software (Dionex Corp.). 2.2.11. Statistical analysis Results from microbiology were expressed as the range of organisms obtained from replicate samples (n ¼ 3). Composition analysis data for polysaccharides, protein, lignin and phenolics are the mean and standard deviation of triplicate samples. A one way analysis of variance was performed to test for compositional differences in BSG between breweries (Neter, Kutner, Nachtsheim, & Wasserman, 1996) and a Student t-test to test for differences in BSG from lager producing and ale-producing breweries. 3. Results and discussion 3.1. Naturally-associated microflora The numbers of each presumptive group of micro-organisms enumerated indicate that the naturally-associated microflora of fresh BSG was predominantly spores of aerobic thermophilic bacteria at numbers between log10 2 and log10 7 g1 depending on the source of the BSG (Table 2). This was as expected since the
Table 3 Proximate analysis of BSG sampled at point of production (mg g1 original dry weight). No.
1 2 3 4 5 6 7 8 9 10 –
Sample code
P01 001a P01 002a P01 003a P01 004a P08 001a P02 001b P05 001b P07 001b P09 001b P11 001b Mean þ/ SD
AIR
796 742 800 816 770 824 798 770 762 705 778 þ/ 36
Polysaccharide
Protein
Klason lignin
Bound phenolics
Mean
SD
Mean
SD
Mean
SD
Mean
SD
433.1 360.5 380.8 417.5 408.6 427.7 355.3 369.9 350.0 344.5 384.8
0.64 64.4 66.7 10.9 8.2 6.5 7.5 11.6 14.3 13.9 33.9
188.1 168.8 103.0 140.9 145.3 222.1 239.7 234.5 214.2 205.2 186.2
3.4 6.5 14.5 8.2 9.2 4.2 4.6 13.7 15.4 11.2 45.4
159.9 132.0 156.8 140.1 127.8 133.9 167.6 125.8 171.5 138.9 145.4
13.8 4.9 24.2 19.7 6.0 2.1 3.6 1.3 31.2 13.6 17.0
8.1 9.2 7.5 10.5 7.9 7.7 9.2 6.5 6.9 7.5 8.1
1.6 2.0 0.5 1.4 0.9 0.2 2.6 0.3 0.2 0.6 1.2
Sample code ¼ unique identifier used for sample traceability to specific brewery. a BSG from ale-producing breweries; b BSG from lager-producing brewery; AIR ¼ alcohol insoluble residue; values for polysaccharide, protein lignin and phenolics are shown as the mean and standard deviation of determinations made in triplicate.
J.A. Robertson et al. / LWT - Food Science and Technology 43 (2010) 890–896 Table 4 Analysis of variance of BSG components.
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Table 6 Analysis of non-cellulosic glucan from BSG (mg g1 original dry sample).
Component
Fexperimental
P
Non-starch polysaccharide Protein Klason lignin Bound phenolics Total starch
0.59 19.17 1.14 0.84 736.79
NS <0.001 NS NS <0.001
Sample code
Analysis of variance of composition data from the 10 breweries. The critical F statistic for p < 0.05 ¼ 2.42. Fexperimental ¼ F statistic calculated from experimental data and P ¼ the corresponding level of statistical significance (NS ¼ not significant: p > 0.05). Non-starch polysaccharides ¼ total polysaccharides determined less the contribution assayed as polymeric starch. Total starch ¼ the contribution from polymeric starch þ maltodextrins present in the BSG.
Total ‘starch’
130 P01 001a 107 P01 002a 74 P01 003a 105 P01 004a P08 001a 72 28 P02 001b b 16 P05 001 b 83 P07 001 b 31 P09 001 21 P11 001b Mean þ/ SD 66.7 þ/ 40.7
Polymeric starch Amylose (%) Mixed linkage glucan 81 nd 63 46 58 25 15 26 nd 22 42.0 þ/ 23.6
46 8 nd 7 63 10 49 11 59 7 57 8 65 4 69 4 nd 5 66 6 59.3 þ/ 8.2 7.0 þ/ 2.4
a
BSG from ale-producing breweries BSG from lager-producing brewery. Total starch ¼ the contribution from polymeric starch þ maltodextrins present in the BSG. Polymeric starch ¼ the starch content determined from AIR analysis. Amylose ¼ the proportion (%) amylose determined in starch from AIR preparations. Nd ¼ not determined. b
temperature used for mashing would select for bacterial spores. The range and variability between breweries also shows that BSG, post-production, is prone to a rapid colonisation by bacteria. The proliferation of anaerobes also indicates a relatively rapid onset of anaerobic conditions post-production of BSG. The apparent proliferation of microaerophilic bacteria and anaerobes in some samples also indicates that the microflora is prone to a rapid change following production of BSG. Although samples were collected on the day of BSG production several hours can elapse between production and processing for microbiology. During this time the bulk in the hot BSG will limit heat loss, encouraging microbial proliferation, and limited access to air will encourage anaerobic conditions to become established. Several micro-organisms, eg Pseudomonas spp., remained below the limit of detection (log102), and numbers of yeasts and moulds were below log104.56. Overall, the profile of the microflora obtained indicates that BSG can be considered microbiologically stable at the point of production but thereafter a rapid change in the microflora can occur, which could promote a rapid deterioration of constituents in BSG. 3.2. Composition 3.2.1. Proximate analysis Fresh BSG samples as provided by the brewery were typically 75– 80% moisture content. Samples P01001 and P11001 were provided dry. The AIR content of the BSG varied between 705 and 820 mg g1 of the sample dry weight (Table 3), which indicates that a significant proportion of the BSG comprises low molecular weight components, removed during preparation of AIR. Polysaccharide was the major component of the BSG, approximately 380 mg g1 (range 345–433),
followed by protein, approximately 190 mg g1 (range 103–240) and Klason lignin, approximately 150 mg g1 (range 126–172). Bound phenolics contributed approximately 10 mg g1 of the BSG dry weight. These components of BSG gave a similar profile to those reported previously (Mandalari et al., 2005). The lipid content and ash content of samples was not measured but typically these components contribute w10% and up to 5% respectively to BSG (Mussatto et al., 2006). There is considerable variation in the composition of BSG between different breweries. Since previous studies (Santos et al., 2003) have shown that replicate samples taken within a brewery were relatively homogeneous, then differences between breweries must be due either to variation in brewery mashing technology or to differences in malt source between breweries. The major malt source used by the breweries was barley base malt. The use of speciality malts and other grain adjuncts cannot be excluded but microscopy showed the BSG samples were apparently exclusively barley-based. An analysis of variance (Table 4) showed that nonstarch polysaccharides (NSP), lignin and bound phenolics in BSG did not vary significantly between breweries but identified the protein content to vary significantly between breweries. Grouping breweries by beer type produced (lager or ale) gave a protein value of 223 mg g1 protein from lager producers whilst that from ale production was 149 mg g1 (p < 0.001). The protein content of lager malts is known to be higher than found in ale malts (Briggs, 1998) and the differences between breweries documented in the current study highlight that different source malt varieties can be an important source of variation in BSG composition. However, since
Table 5 Component sugars determined in AIR preparations from BSG (mg g1 AIR). Sample
Rha
Fuc
Ara
Xyl
Man
Gal
Glc
UA
Total Mean
P01 001a P01 002a P01 003a P01 004a P08 001a P02 001b P05 001b P07 001b P09 001b P11 001b Mean þ/ SD
1.4 1.4 2.6 1.7 1.4 0.8 3.1 0.9 1.3 1.2 1.6 þ/ 0.7
2.3 1.8 0.8 2.8 1.2 0.3 1.7 0.3 0.9 1.4 1.3 þ/ 0.8
76.4 76.9 73.2 78.9 79.2 92.4 83.4 79.4 74.1 86.9 80.1 þ/ 6.0
185.7 160.0 150.1 165.1 144.3 201.9 150.3 151.5 169.6 159.8 163.8 þ/ 17.9
7.5 6.6 5.9 7.1 9.0 5.2 7.5 5.5 5.5 6.8 6.7 þ/ 1.2
11.9 10.7 8.6 11.6 9.6 11.8 11.5 10.7 8.6 11.1 10.6 þ/ 1.3
Values are the mean of determinations made in triplicate. UA ¼ uronic acid. A:X ¼ ratio ara:xyl. a BSG from ale-producing breweries b BSG from lager-producing brewery
204.2 181.7 203.1 197.4 238.3 206.1 137.0 181.1 173.4 174.2 189.7 þ/ 26.9
54.7 46.7 31.7 47.2 47.9 38.5 50.9 51.0 25.9 47.3 44.2 þ/ 9.2
A:X SD
544.1 0.8 485.8 86.8 476.0 43.3 511.7 13.3 530.7 10.7 557.0 7.9 445.3 9.4 480.4 15.1 459.3 18.8 488.6 19.7 497.9 þ/ 36.8
0.41 0.48 0.49 0.48 0.54 0.46 0.55 0.52 0.44 0.54 0.50 þ/ 0.05
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J.A. Robertson et al. / LWT - Food Science and Technology 43 (2010) 890–896
Table 7 Component alkali-extractable phenolic acids determined in AIR preparations from BSG (mg g1 AIR). Sample code a
P01 001 P01 002a P01 003a P01 004a P08 001a P02 001b P05 001b P07 001b P09 001b P11 001b Mean þ/ SD a b
Coumaric Acid
8,80 ; 6,70 -DiFA
Ferulic acid
8,80 -DiFA
8,50 -DiFA
5,50 -DiFA
8-0-40 -DiFA
8,50 ; 7-O-40 -DiFA
Total þ/ SD
3.2 2.0 1.4 2.1 1.7 2.0 1.8 1.3 1.5 1.4 1.8 þ/ 0.6
0.4 0.8 0.8 0.1 0.6 0.6 0.9 0.5 1.0 0.9 0.7 þ/ 0.3
3.9 6.3 4.4 6.7 5.2 3.8 6.0 3.9 3.9 5.3 4.9 þ/ 3.0
0.0 0.1 0.1 0.5 0.1 0.1 0.1 0.1 0.1 0.4 0.2 þ/ 0.2
0.2 0.4 0.5 0.5 0.2 0.4 0.0 0.4 0.6 0.0 0.3 þ/ 0.2
0.5 0.9 0.5 0.9 0.7 0.6 0.9 0.5 0.5 0.9 0.7 þ/ 0.2
1.6 1.7 1.2 2.0 1.4 1.5 1.4 1.3 1.3 1.3 1.5 þ/ 0.2
0.0 0.0 0.1 0.0 0.1 0.3 0.0 0.2 0.1 0.2 0.1 þ/ 0.1
10.22.0 12.42.6 9.40.7 12.91.7 10.20.3 9.40.3 11.53.3 8.40.4 9.11.2 10.60.8 10.4 þ/ 1.5
BSG from ale-producing breweries BSG from lager-producing brewery. DiFA ¼ diferulic acid
the difference in BSG is readily distinguished by beer type produced then this can be taken into account when selecting BSG for downstream processing. 3.2.2. Polysaccharides Polysaccharides, as NSP and starch, accounted for approximately 50% of the AIR in BSG. From the predominance of arabinose, xylose and glucose (Table 5) the major polysaccharides were inferred to be arabinoxylan (AX) and glucan. The ara:xyl ratio for all samples (0.50 þ/ 0.05) was similar and similar to previously reported ratios (Mandalari et al., 2005), suggesting that malt source has little effect on AX composition in BSG. The low content of galactose (Table 5) indicates that arabinogalactans (AG) (Loosveld, Grobet, & Delcour, 1997) are a minor component of the BSG. Cellulose, estimated as the difference between total glucan and polymeric starch was 19.7% þ/ 2.7 of the original BSG dry weight. This estimate also includes a contribution from the low levels of mixed-linkage present in the glucan, (w1%: see below), and compares with a reported cellulose content of BSG w17%, (Mussatto et al., 2006; Valverde, 1994). Detailed analysis for total starch-based material and mixedlinkage b-glucans (Table 6) indeed showed BSG to contain a variable but significant amount of residual starch (16–130 mg g1). BSG from lager producers contained 35.8 mg g1 starch whilst from ale producers this was 97.6 mg g1 starch. Whilst overlap exists in the starch content of BSG from breweries producing either ale or lager, an analysis of variance (Table 4) showed there was a significant
difference (p < 0.001) between beer types. Starch analysis on the AIR compared to the BSG prior to AIR preparation showed that a proportion of the total starch could be accounted for as lower molecular weight (alcohol soluble) maltodextrins. These maltodextrins represented approximately 37% of the total starch measured and this proportion was similar for all breweries. At a ratio of w60% amylose in the residual starch, this amylose content was higher than expected for barley starch (w25% proportion of amylose) (Tester, 1997; You & Izydorczyk, 2002). Although this suggests there may be some resistance to digestion of amylose during mashing, the levels of residual starch indicate there is little apparent effect on the overall digestion of starch either between malt types or between breweries. Mixed linkage b-glucan in BSG, 7 mg g1 dry weight, was similar for all breweries. The much reduced content of mixed-linkage b-glucan in BSG compared to barley grain (25–54 mg g1) and malt (Manzanares & Sendra, 1996; Papageorgiou, Lakhdara, Lazaridou, Biliaderis, and Izydorczyk (2005); Gamlath, Aldreda, & Panozzo, 2008) demonstrates an extensive degradation of mixed-linkage b-glucan during mashing and hence that BSG is unsuitable for exploitation as a source of mixed-linkage b-glucan in functional foods. 3.2.3. Cell wall-bound phenolics Bound phenolics are a minor component in BSG AIR (Table 7) and predominantly based on ferulic acid and its dehydrodimers. The spectrum and concentration of bound phenolics is also similar
tryptophan arginine lysine histidine phenylalanine tyrosine leucine isoleucine valine glycine alanine proline glutamic acid / glutamine aspartic acid / asparagine serine threonine hydroxproline methionine cysteine 0
5
10
15
20
25
Amino Acid (g/100g amino acid) Fig. 1. Profile of amino acid determined in BSG samples. Samples, in triplicate, corresponding to 10 mg nitrogen (N) were hydrolysed with 6 M HCl for 23 hours at pH 2.2. Hydrolysates were analysed on a Biochrom 30 Amino Acid Analyser, except for tryptophan, which was analysed by HPLC. (black) ¼ ale; (grey) ¼ lager. The values for ale correspond to samples 1 – 5 (Table 3) and for lager to samples 6 – 10 (Table 3). Values are shown as the mean þ 1sd.
J.A. Robertson et al. / LWT - Food Science and Technology 43 (2010) 890–896
for all samples. On the basis of the overall arabinose content and ferulate-based components in BSG then an estimated 1 in 12 arabinose units is esterified to a ferulate. Also, around 60% of the esterified ferulate is estimated to be present as ferulic acid, with the remaining 40% involved in dehydrodimer crosslinking. When crosslinking is expressed as the ratio of xylose to dehydrodimers (Antoine et al., 2003), the gross index of cross linking for BSG is w52. This shows BSG to be a highly crosslinked material, much more so than wheat bran (Antoine et al., 2003). Overcoming the crosslinking to control the release of component polysaccharides and protein is seen as a major challenge to the exploitation of BSG (Robertson, Faulds, Smith, & Waldron, 2008). 3.2.4. Protein and amino acids Variation in protein content of BSG between breweries (Table 4) was ascribed to the malt/beer source, with BSG from lager production having a significantly higher protein content (223.1 mg g1 þ/ 14.2) compared to BSG from ale production (149.2 mg g1 þ/ 32.1). However, amino acid analysis (Fig. 1) gave a similar profile of amino acids for ale and lager sourced BSG. The predominant amino acid was glutamine/glutamic acid, as expected since the glutamine-rich barley storage proteins, hordeins, are a major component of the protein present in BSG. During mashing, hordeins are involved in extensive disulphide bond formation, resulting in an aggregate formation and reduction in solubility (Celus et al., 2006). Aggregate formation would account for the persistence of hordeins in AIR preparations and their presence may also form a constraint on the solubilisation of components within the BSG matrix; as suggested by a recent study showing that disruption of the protein matrix by peptidases enhances the effectiveness of glycoside hydrolases and esterase on BSG (Faulds et al., 2009). On the other hand, it remains fully possible to solubilize most of the protein in BSG using a mild alkaline extraction and/or enzymatic methods (Celus, Brijs, & Delcour, 2007; Treimo, Aspmo, Eijsink, & Horn, 2008). 4. Conclusion The analysis of fresh samples of BSG obtained on the day of production, from a range of breweries, has shown that BSG from lager brewing has a higher protein content but lower residual starch content than BSG from ale brewing. These differences are consistent with the different source malts. In general, the nonstarch polysaccharide profile of BSG is a relatively uniform between breweries, as is the protein composition. Lignin content and cell wall bound phenolics also show little variation between breweries. Thus, as a feedstock available for industrial upgrading differences between beer type brewed is the major source variation. Since differences in protein and starch content can be identified from the type of beer being produced this should not present problems with a routine industrial upgrading of BSG. However, the high moisture content and composition of BSG makes it highly susceptible to microbial degradation. It is the presence of a resident microflora which is most likely to affect the potential to use BSG as a reliable and uniform industrial feedstock for value-added downstream processing. However, at the point of production, microbial contamination is low and considered within acceptable limits for food use, but appropriate storage regimens will be required to minimise microbial proliferation prior to downstream processing of the BSG. Acknowledgements We gratefully acknowledge funding from the Biotechnology and Biological Sciences Research Council (BBSRC) UK, and the
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Commission of the European Communities contract FOOD-CT2005-006922 ‘‘Reducing Food Processing Waste’’. This study does not necessarily reflect the views of the commission and its future policy in this area. References Adler-Nissen, J. (1986). Enzymic hydrolysis of food proteins. London: Elsevier Applied Science Publishers. Antoine, C., Peyron, S., Mabille, F., Lapierre, C., Bouchet, B., Abecassis, J., et al. (2003). Individual contribution of grain outer layers and their cell wall structure to the mechanical properties of wheat bran. Journal of Agricultural and Food Chemistry, 51, 2026–2033. Blakeney, A. B., Harris, P. J., Henry, R. J., & Stone, B. A. (1983). A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohydrate Research, 113, 291–299. Blumenkranz, N., & Asboe-Hansen, G. (1973). New method for quantitative determination of uronic acids. Analytical Biochemistry, 54, 484–489. Briggs, D. E. (1998). Section 6.9, the nitrogen or protein content of barley. In. Malts and malting. Chapter 6. The selection and purchase of grain. Berlin, Heidelberg, New York: Springer-Verlag. Celus, I., Brijs, K., & Delcour, J. A. (2006). The effects of malting and mashing on barley protein extractability. Journal of Cereal Science, 44, 203–211. Celus, I., Brijs, K., & Delcour, J. A. (2007). Enzymatic hydrolysis of brewers’ spent grain proteins and technofunctional properties of the resulting hydrolysates. Journal of Agricultural and Food Chemistry, 55, 8703–8710. Dubois, M., Gilles, K., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356. EC, Commission Directive 98/64/EC of 3 September1998. (1998). Part A, determination of amino acids. Official Journal of the European Communities, L 257, 14–28. EC, Commission Directive 2000/45/EC of 6 July 2000. (2000). Part C, determination of tryptophan. Official Journal of the European Communities, L174, 32–50. Faulds, C. B., Collins, S., Robertson, J. A., Treimo, J., Eijsink, V. G. H., Hinz, S. W. A., Schols, H. A., Buchert, J., & Waldron, K. W. (2009). Protease-induced solubilisation of carbohydrates from brewers’ spent grain. Journal of Cereal Science, 50, 332–336. Forssell, P., Kontkanen, H., Schols, H. A., Hinz, S., Eijsink, V. G. H., Treimo, J., et al. (2008). Hydrolysis of brewers’ spent grain by carbohydrate degrading enzymes. Journal of the Institute of Brewing, 114, 306–314. Gamlath, J., Aldreda, G. P., & Panozzo, J. F. (2008). Barley (1-3;1-4)-b-glucan and arabinoxylan content are related to kernel hardness and water uptake. Journal of Cereal Science, 47, 365–371. ICMSF. (1978). Microorganisms in foods. 1. Their significance and methods of enumeration. International Commission for the Microbiological Specifications for Foods (2nd ed.). Toronto, Canada: University of Toronto Press. Jones, D. B. (1941). Factors for converting percentages of nitrogen in foods and feeds into percentages of proteins. United States Department of Agriculture, 183, 1–22. Loosveld, A.-M. A., Grobet, P. J., & Delcour, J. A. (1997). Contents and structural features of water-extractable arabinogalactan in wheat flour fractions. Journal of Agriculture & Food Chemistry., 45, 1998–2002. Mandalari, G., Faulds, C. B., Sancho, A. I., Saija, A., Bisignano, G., LoCurto, R., et al. (2005). Fractionation and characterisation of arabinoxylans from brewers’ spent grain and wheat bran. Journal of Cereal Science, 42, 205–212. Manzanares, P., & Sendra, J. M. (1996). Determination of total (1 / 3),(1 / 4)-b-Dglucan in barley and malt flour samples. Journal of Cereal Science, 23, 293–296. McCleary, B. V., Gibson, T. S., & Mugford, D. C. (1997). Measurement of total starch in cereal products by amyloglucosidase-alpha-amylase method: collaborative study. Journal of AOAC International, 80, 571–579. Mussatto, S. I., Dragone, G., & Roberto, I. C. (2006). Brewers’ spent grain: generation, characteristics and potential applications. Journal of Cereal Science, 43, 1–14. Neter, J., Kutner, M., Nachtsheim, C., & Wasserman, W. (1996). Sections 16.8 and 16.9. In. Applied linear statistical models. Boston, MA: The McGraw-Hill Companies, Inc. Palmer, J. J. (2006). Chapter 12: what is malted grain?. In. How to brew Boulder, Colorado, USA: Brewers Association. Papageorgiou, M., Lakhdara, N., Lazaridou, A., Biliaderis, C. G., & Izydorczyk, M. S. (2005). Water extractable (1 / 3),(1 / 4)-b-D-glucans from barley and oats: an intervarietal study on their structural features and rheological behaviour. Journal of Cereal Science, 42, 213–224. Robertson, J. A., Faulds, C. B., Smith, A. C., & Waldron, K. W. (2008). Peroxidasemediated oxidative cross-linking and its potential to modify mechanical properties in water-soluble polysaccharide extracts and cereal grain residues. Journal of Agricultural and Food Chemistry, 56, 1720–1726. Santos, M., Jime´nez, J. J., Bartolome´, B., Go´mez-Cordove´s, C., & del Nozal, M. J. (2003). Variability of brewer’s spent grain within a brewery. Food Chemistry, 80, 17–21. Tester, R. F. (1997). Influence of growth conditions on barley starch properties. International Journal of Biological Macromolecules, 21, 37–45. Townsley, P. M. (1979). Preparation of commercial products from brewer’s waste grain and trub. MBAA Technical Quarterly, 16, 130–134. Treimo, J., Aspmo, S. I., Eijsink, V. G. H., & Horn, S. J. (2008). Enzymatic solubilization of proteins in brewer’s spent grain. Journal of Agricultural and Food Chemistry, 56, 5359–5365.
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Treimo, J., Westereng, B., Horn, S. J., Forssell, P., Robertson, J. A., Faulds, C. B., et al. (2009). Enzymatic solubilization of brewers’ spent grain by combined action of carbohydrases and peptidases. Journal of Agricultural and Food Chemistry, 57, 3316–3324. Valverde, P. (1994). Barley spent grain and its future. Cerveza y Malta, 122, 7–26.
Waldron, K. W., Parr, A. J., Ng, A., & Ralph, J. (1996). Cell wall esterified phenolic dimers: identification and quantification by reverse phase high performance liquid chromatography and diode array detection. Phytochemical Analysis, 7, 305–312. You, S. G., & Izydorczyk, M. S. (2002). Molecular characteristics of barley starches with variable amylose content. Carbohydrate Polymers, 49, 33–42.
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Profiling brewers’ spent grain for composition and microbial ecology at the site of production James A. Robertson a, *, Kerry J.A. I’Anson b, Janneke Treimo c, Craig B. Faulds a, Tim F. Brocklehurst b, Vincent G.H. Eijsink c, Keith W. Waldron a a b c
Sustainable Food Chain Exploitation Platform, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom Microbial Ecology Exploitation Platform, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, N-1432 Aas, Norway
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2009 Received in revised form 14 January 2010 Accepted 19 January 2010
Brewers’ spent grain (BSG) is a readily available, high volume low cost byproduct of brewing and is a potentially valuable resource for industrial exploitation. The variation in BSG composition and the implications for microbiological spoilage by a resident microflora might affect the potential to use BSG as a reliable food-grade industrial feedstock for value-added downstream processing. Fresh samples of BSG from a range of 10 breweries have been analysed for their microbial and chemical composition. The results show that a resident microflora of mainly thermophilic aerobic bacteria (<107 g-1 fresh weight) persists on BSG. This population is susceptible to rapid change but at the point of production BSG can be considered microbiologically stable. Chemically, BSG is rich in polysaccharides, protein and lignin. Residual starch can contribute up to 13% of the dry weight and BSG from lager malts has higher protein content than that from ale. In general, at the point of production, BSG is a relatively uniform chemical feedstock available for industrial upgrading. Differences between breweries should not present problems when considering BSG for industrial exploitation but susceptibility to microbial colonisation is identified as a potential problem area which might restrict its successful exploitation. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Keywords: Brewers’spent grain Barley Microbiology Polysaccharide Phenolic Protein
1. Introduction Brewers’ spent grain (BSG) is obtained from barley (Hordeum vulgare L.) as the outer pericarp-seed coat layers from the original malted barley grain which remain after hot water extraction at 65– 70 C (mashing) (Mussatto, Dragone, & Roberto, 2006). It can represent approximately 30% (w/w) of the starting malted grain (Townsley, 1979), which makes BSG a readily available, high volume and low cost byproduct within the brewing industry. Although difficulties persist in the extraction of component polysaccharides and proteins (Celus, Brijs, & Delcour, 2006; Forssell et al., 2008; Treimo et al., 2009) BSG remains a potentially more valuable resource for industrial exploitation than the current general use as an animal feed. Indeed, value-added products are increasingly being sought from BSG but information on microbiological safety and its uniformity for use as an industrial feedstock can limit its appeal for exploitation.
* Corresponding author. Tel.: þ44 1603 255000; fax: þ44 1603 507723. E-mail address: [email protected] (J.A. Robertson).
Whilst the gross composition of BSG samples has been well documented (Mussatto et al., 2006; Santos, Jime´nez, Bartolome´, Go´mez-Cordove´s, & del Nozal, 2003; Valverde, 1994) less is known about the expected variation in the composition of BSG between breweries and the extent of its microbial contamination on production. Certainly the rich polysaccharide and protein content and the high moisture content of BSG makes it particularly susceptible to microbial growth and subsequent spoilage (Valverde, 1994). Where storage may be required for downstream processing of BSG then deterioration through microbial activity is perceived as a potential problem, unless the BSG can be stabilised post-production. Variation in BSG composition can be expected to arise from differences in barley malting cultivars, malting practices, adjuncts added and wort production during mashing processes in breweries. A major difference is that different barley cultivars are used as the malt source for lager and ale. Also, in general ale malt is kilned at a higher temperature (Palmer, 2006) whilst lager malts are derived from barley with a higher protein content (Briggs, 1998). Spent grain is often mixed with yeast, removed post-fermentation, and cold break protein (trub) separated from wort. Samples provided by breweries were obtained directly from the mash tun and hence the effects of yeast or trub was not considered.
0023-6438/$ – see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2010.01.019
J.A. Robertson et al. / LWT - Food Science and Technology 43 (2010) 890–896
In the current study the composition and resident microflora of BSG samples, obtained on the day of production, from a range of 10 breweries have been screened to estimate variation between breweries. This complements a previous study, where estimation of variation was limited to a within brewery comparison on composition (Santos et al., 2003). The results from the current study indicate to what extent BSG can be considered a reliable and uniform industrial feedstock for value-added downstream processing. 2. Materials and methods 2.1. Materials BSG from a range of 10 commercial breweries throughout the EU and South Africa was obtained as a single bulk sample from each brewery. Samples were the residues resulting from wort prepared from malted barley, for either lager or ale production. The BSG was supplied fresh from the mash tun, chilled to w4 C to facilitate microbiological analysis, unless otherwise stated. Samples of the BSG supplied were also freeze dried and milled to pass a 0.5 mm sieve prior to use for experimental analysis. All chemicals used were at least Analar quality. 2.2. Methods 2.2.1. Microbiology The naturally-associated microflora was enumerated using methods described by the International Commission on the Microbiological Specifications for Foods (ICMSF, 1978). In summary, within 24 h of production and delivery to the laboratory, samples of BSG (40 g fresh weight) were blended with peptone salt dilution fluid (PSDF) (360 mL) in a Stomacher Lab-blender 400 (Seward Laboratory) for 2 min. Viable micro-organisms from each sample were enumerated by plating serial dilutions of each supernatant, made in PSDF, onto the surface of a range of culture media using a Spiral Plate Maker. The culture media, incubation conditions, and the presumptive identification of micro-organisms enumerated on each medium were as shown in Table 1. Where further refinement of the presumptive identification was required, bacteria were subjected to standard Gram’s stain, and oxidase and catalase tests. 2.2.2. Moisture determination Samples of fresh BSG were dried to constant weight (16 h) at 104 C in a forced draught oven to determine dry weight/g fresh weight. 2.2.3. Alcohol-insoluble residues (AIR) preparation AIR were prepared prior to analysis for cell wall sugars, lignin, cell wall bound phenolic acids, starch and mixed-linkage b-glucans. Wet BSG (100 g; w22% dry weight; stored up to 24 h at 4 C) was Table 1 Presumptive groups of microflora enumerated from BSG. Presumptive Group
Plating medium
Aerobic mesophilic Plate Count Agar (PCA) bacteria Aerobic thermophilic Plate Count Agar (PCA) bacteria Pseudomonas spp. Cephaloridine, Fucidin, Cetrimide Agar (CFC) Yeasts and moulds Oxytetracycline, Dextrose, Yeast Extract Agar (ODY) Microaerophilic De Man, Rogosa, Sharpe bacteria Agar (MRS) Strictly anaerobic Oxygen-free Reinforced bacteria Clostridial Medium (RCM)
Incubation conditions In air at 20 C In air at 55 C In air at 25 C In air at 20 C Gaseous atmosphere of 90% H2 and 10% CO2 at 25 C Gaseous atmosphere of 90% H2 and 10% CO2 at 25 C
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homogenised for 30 s then plunged into boiling absolute ethanol (300 mL: w80% ethanol final concentration) for 1 h. The insoluble residue was recovered by filtration (GF/C paper using a Buchner funnel). After a further 2 extractions in boiling 70% ethanol (v/v) (each 300 mL; 2 h) the residue was extracted (2) in boiling absolute ethanol (300 mL; 5 min), then washed with cold absolute ethanol (150 mL). An aliquot of the final filtrate was tested for residual non-cell wall soluble sugars using phenol-sulphuric acid (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), to give a final filtrate containing less than 10 mg mL1 sugar. The insoluble residue (AIR) was washed in 2 volumes acetone (2), with washings removed by gentle suction, prior to drying to constant weight at 40 C. Except for protein analysis and starch maltodextrins all chemical analyses were performed on AIRs. 2.2.4. Sugars determination Sugars were determined as total sugars released from the sample AIR through Saeman hydrolysis, (pre-hydrolysis using 12 M H2SO4: 3 h room temperature followed by dilution to 1 M H2SO4) and hydrolysis for 1 h at 105 C). Hydrolysates were derivatised as their alditol acetates, and analysed by GLC using a flame ionisation detector (Blakeney, Harris, Henry, & Stone, 1983). Total uronic acid content was determined photometrically using glucuronic acid as a standard (Blumenkranz & Asboe-Hansen, 1973). 2.2.5. Klason lignin determination Lignin was determined as Klason lignin. Samples (w50 mg) were dispersed in 0.75 mL of 12 M sulphuric acid and incubated at room temperature (3 h) prior to dilution to 1 M acid concentration. After a further incubation (100 C for 2.5 h in sealed tubes) the insoluble residue was recovered by filtration (No. 3 glass sinter) under gentle suction and washed free of acid. Klason lignin was determined as the weight gain in each glass sinter after drying at 50 C to constant weight in a temperature controlled oven. 2.2.6. Cell wall bound phenolic acids determination Total alkali-extractable phenolic acid present in the samples were determined following saponification with 4 M NaOH (w5 mg mL1; 18 h; room temperature in the dark). Supernatants were recovered by centrifugation. An aliquot (0.8 mL) was acidified (pH w 2) using concentrated HCl, prior to extraction with ethyl acetate (3 3 mL). Extracts were reduced to dryness under nitrogen, resuspended in methanol: H2O (50/50, v/v) and constituent alkali soluble phenolic acids were separated and quantified using HPLC (LUNA C18 reverse phase HPLC column (Phenonomex, Macclesfield, UK)) according to the method of Waldron, Parr, Ng, and Ralph (1996), using trans-cinnamic acid as internal standard. 2.2.7. Starch determination Starch was determined as total starch (K-TSTA: Megazyme International Ireland Ltd.) based on the method of McCleary, Gibson, & Mugford, 1997. In brief, starch was measured as glucose following sample dispersion in DMSO, digestion using a thermostable a-amylase, followed by amyloglucosidase treatment and photometric quantification of glucose released. The assay conditions for starch assumed a starch content <100 mg g1 BSG in a10 mL reaction volume. Freeze dried samples were used to determine total starch, as starch þ dextrins, and AIR to distinguish polymeric starch. Amylose was determined using Megazyme kit AM/AMP 01/96. Samples were dispersed in DMSO and starch recovered through ethanol precipitation. After redissolution, amylopectin was specifically removed from solution using Con A and the supernatant was analysed for starch (amylose) through digestion with amylase/amyloglucosidase and quantification of glucose released.
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Table 2 Microbiological characterization of brewers’ spent grain samples. Log10 micro-organisms/g fresh weight brewers’ spent grain
Media/Micro-organisms
Sample code
PCA 20 C Aerobic Mesophilic bacteria PCA 55 C Aerobic Thermophilic bacteria CFC 25 C Pseudomonas spp. bacteria ODY 20 C Yeasts & moulds bacteria MRS 25 C Microaerophilic bacteria RCM 25 C Anaerobic bacteria
P01 004
P01 003
P02 001
P05 001
P08 001
P09 001
P11 001
2.58 3.06 <2 <2 <2 <2
2.90–5.33 5.33–6.60 <2 2.66–3.18 <2 2.43–4.76
3.59–4.43 6.00–7.00 <2 3.45–4.56 7.84–7.93 5.58–6.14
3.00–4.70 2.25–4.00 <2 <2 <2 1.95–3.00
3.10–3.60 2.00–2.30 <2 <2 <2 <2
5.90–6.12 3.96–5.30 <2 1.96–2.20 >7 >7
2.60–3.20 2.60–3.20 <2 <2 2.40–2.50 2.50–3.30
Sample code ¼ unique identifier used for sample traceability to specific brewery
2.2.8. Mixed-linkage b-glucan determination Mixed-linkage b-glucan, [(1-3),(1-4)-b-D-glucan], was assayed through selective degradation of the (1-3) linkage using lichenase (a specific endo-(1-3),(1-4)-b-D-glucan 4-glucanohydrolase) followed by release of glucose, using b-glucosidase, and its photometric assay using glucose oxidase, (K-BGLU: Megazyme International Ireland Ltd.). The kit procedure, based on 1 g sample weight was modified for use with a 100 mg sample weight. 2.2.9. Total nitrogen determination Nitrogen content of BSG was quantified using the Kjeldahl method. The samples were prepared and measured on a Kjeltec Auto 1035 sampler system (Tecator, Nerliens, Oslo, Norway) as described by Adler-Nissen (1986). Protein content was calculated from nitrogen using a conversion factor of 6.25 (Jones, 1941). 2.2.10. Amino acid composition analysis The amino acid composition of freeze dried BSG analyses was determined by the Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences according to standard EU methods (EC, Commission Directive 98/64/EC, 1998; EC, Commission Directive 2000/45/EC, 2000). In brief, a sample quantity corresponding to 10 mg nitrogen (N) was treated with a performic acid/phenol mixture to oxidise S-containing amino acids, hydrolysed with 6 M HCl for 23 h, pH-adjusted to 2.2, diluted with 0.2 M sodium citrate loading buffer, pH 2.2 (Biochrom Ltd., Cambridge, UK) and micro-filtrated (0.45 mm Spartan membrane filter, Schleicher & Schuell, Dassel, Germany) prior to analysis on a Biochrom 30 Amino Acid Analyser (Biochrom Ltd.) equipped with an Oxidized Feedstuff High Performance PEEK Column (Biochrom Ltd.). Amino acids were detected and quantified photometrically at 440 and 570 nm after post-column
derivatisation with ninhydrin. Data were analysed against appropriate external standards (Sigma Chemical, St. Louis, Mo., U.S.A.) using the ChromeleonÒ Chromatography Management Software (Dionex Corporation, Sunnyvale, CA). Tryptophan was analysed by HPLC (Summit HPLC System; Dionex Corp.), equipped with a SUPELCOSILÔ LC-18 column (Supelco, Bellefonte, Pa, USA) and a fluorescence detector (Shimadzu RF – 535, Shimadzu Corporation, Kyoto, Japan), after alkaline hydrolysis (EC, Commission Directive 2000/45/EC, 2000). Data were analysed against appropriate external standards (DL-Tryptophan and alphamethyl-tryptophan; Sigma Chemical) using the ChromeleonÒ Chromatography Management Software (Dionex Corp.). 2.2.11. Statistical analysis Results from microbiology were expressed as the range of organisms obtained from replicate samples (n ¼ 3). Composition analysis data for polysaccharides, protein, lignin and phenolics are the mean and standard deviation of triplicate samples. A one way analysis of variance was performed to test for compositional differences in BSG between breweries (Neter, Kutner, Nachtsheim, & Wasserman, 1996) and a Student t-test to test for differences in BSG from lager producing and ale-producing breweries. 3. Results and discussion 3.1. Naturally-associated microflora The numbers of each presumptive group of micro-organisms enumerated indicate that the naturally-associated microflora of fresh BSG was predominantly spores of aerobic thermophilic bacteria at numbers between log10 2 and log10 7 g1 depending on the source of the BSG (Table 2). This was as expected since the
Table 3 Proximate analysis of BSG sampled at point of production (mg g1 original dry weight). No.
1 2 3 4 5 6 7 8 9 10 –
Sample code
P01 001a P01 002a P01 003a P01 004a P08 001a P02 001b P05 001b P07 001b P09 001b P11 001b Mean þ/ SD
AIR
796 742 800 816 770 824 798 770 762 705 778 þ/ 36
Polysaccharide
Protein
Klason lignin
Bound phenolics
Mean
SD
Mean
SD
Mean
SD
Mean
SD
433.1 360.5 380.8 417.5 408.6 427.7 355.3 369.9 350.0 344.5 384.8
0.64 64.4 66.7 10.9 8.2 6.5 7.5 11.6 14.3 13.9 33.9
188.1 168.8 103.0 140.9 145.3 222.1 239.7 234.5 214.2 205.2 186.2
3.4 6.5 14.5 8.2 9.2 4.2 4.6 13.7 15.4 11.2 45.4
159.9 132.0 156.8 140.1 127.8 133.9 167.6 125.8 171.5 138.9 145.4
13.8 4.9 24.2 19.7 6.0 2.1 3.6 1.3 31.2 13.6 17.0
8.1 9.2 7.5 10.5 7.9 7.7 9.2 6.5 6.9 7.5 8.1
1.6 2.0 0.5 1.4 0.9 0.2 2.6 0.3 0.2 0.6 1.2
Sample code ¼ unique identifier used for sample traceability to specific brewery. a BSG from ale-producing breweries; b BSG from lager-producing brewery; AIR ¼ alcohol insoluble residue; values for polysaccharide, protein lignin and phenolics are shown as the mean and standard deviation of determinations made in triplicate.
J.A. Robertson et al. / LWT - Food Science and Technology 43 (2010) 890–896 Table 4 Analysis of variance of BSG components.
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Table 6 Analysis of non-cellulosic glucan from BSG (mg g1 original dry sample).
Component
Fexperimental
P
Non-starch polysaccharide Protein Klason lignin Bound phenolics Total starch
0.59 19.17 1.14 0.84 736.79
NS <0.001 NS NS <0.001
Sample code
Analysis of variance of composition data from the 10 breweries. The critical F statistic for p < 0.05 ¼ 2.42. Fexperimental ¼ F statistic calculated from experimental data and P ¼ the corresponding level of statistical significance (NS ¼ not significant: p > 0.05). Non-starch polysaccharides ¼ total polysaccharides determined less the contribution assayed as polymeric starch. Total starch ¼ the contribution from polymeric starch þ maltodextrins present in the BSG.
Total ‘starch’
130 P01 001a 107 P01 002a 74 P01 003a 105 P01 004a P08 001a 72 28 P02 001b b 16 P05 001 b 83 P07 001 b 31 P09 001 21 P11 001b Mean þ/ SD 66.7 þ/ 40.7
Polymeric starch Amylose (%) Mixed linkage glucan 81 nd 63 46 58 25 15 26 nd 22 42.0 þ/ 23.6
46 8 nd 7 63 10 49 11 59 7 57 8 65 4 69 4 nd 5 66 6 59.3 þ/ 8.2 7.0 þ/ 2.4
a
BSG from ale-producing breweries BSG from lager-producing brewery. Total starch ¼ the contribution from polymeric starch þ maltodextrins present in the BSG. Polymeric starch ¼ the starch content determined from AIR analysis. Amylose ¼ the proportion (%) amylose determined in starch from AIR preparations. Nd ¼ not determined. b
temperature used for mashing would select for bacterial spores. The range and variability between breweries also shows that BSG, post-production, is prone to a rapid colonisation by bacteria. The proliferation of anaerobes also indicates a relatively rapid onset of anaerobic conditions post-production of BSG. The apparent proliferation of microaerophilic bacteria and anaerobes in some samples also indicates that the microflora is prone to a rapid change following production of BSG. Although samples were collected on the day of BSG production several hours can elapse between production and processing for microbiology. During this time the bulk in the hot BSG will limit heat loss, encouraging microbial proliferation, and limited access to air will encourage anaerobic conditions to become established. Several micro-organisms, eg Pseudomonas spp., remained below the limit of detection (log102), and numbers of yeasts and moulds were below log104.56. Overall, the profile of the microflora obtained indicates that BSG can be considered microbiologically stable at the point of production but thereafter a rapid change in the microflora can occur, which could promote a rapid deterioration of constituents in BSG. 3.2. Composition 3.2.1. Proximate analysis Fresh BSG samples as provided by the brewery were typically 75– 80% moisture content. Samples P01001 and P11001 were provided dry. The AIR content of the BSG varied between 705 and 820 mg g1 of the sample dry weight (Table 3), which indicates that a significant proportion of the BSG comprises low molecular weight components, removed during preparation of AIR. Polysaccharide was the major component of the BSG, approximately 380 mg g1 (range 345–433),
followed by protein, approximately 190 mg g1 (range 103–240) and Klason lignin, approximately 150 mg g1 (range 126–172). Bound phenolics contributed approximately 10 mg g1 of the BSG dry weight. These components of BSG gave a similar profile to those reported previously (Mandalari et al., 2005). The lipid content and ash content of samples was not measured but typically these components contribute w10% and up to 5% respectively to BSG (Mussatto et al., 2006). There is considerable variation in the composition of BSG between different breweries. Since previous studies (Santos et al., 2003) have shown that replicate samples taken within a brewery were relatively homogeneous, then differences between breweries must be due either to variation in brewery mashing technology or to differences in malt source between breweries. The major malt source used by the breweries was barley base malt. The use of speciality malts and other grain adjuncts cannot be excluded but microscopy showed the BSG samples were apparently exclusively barley-based. An analysis of variance (Table 4) showed that nonstarch polysaccharides (NSP), lignin and bound phenolics in BSG did not vary significantly between breweries but identified the protein content to vary significantly between breweries. Grouping breweries by beer type produced (lager or ale) gave a protein value of 223 mg g1 protein from lager producers whilst that from ale production was 149 mg g1 (p < 0.001). The protein content of lager malts is known to be higher than found in ale malts (Briggs, 1998) and the differences between breweries documented in the current study highlight that different source malt varieties can be an important source of variation in BSG composition. However, since
Table 5 Component sugars determined in AIR preparations from BSG (mg g1 AIR). Sample
Rha
Fuc
Ara
Xyl
Man
Gal
Glc
UA
Total Mean
P01 001a P01 002a P01 003a P01 004a P08 001a P02 001b P05 001b P07 001b P09 001b P11 001b Mean þ/ SD
1.4 1.4 2.6 1.7 1.4 0.8 3.1 0.9 1.3 1.2 1.6 þ/ 0.7
2.3 1.8 0.8 2.8 1.2 0.3 1.7 0.3 0.9 1.4 1.3 þ/ 0.8
76.4 76.9 73.2 78.9 79.2 92.4 83.4 79.4 74.1 86.9 80.1 þ/ 6.0
185.7 160.0 150.1 165.1 144.3 201.9 150.3 151.5 169.6 159.8 163.8 þ/ 17.9
7.5 6.6 5.9 7.1 9.0 5.2 7.5 5.5 5.5 6.8 6.7 þ/ 1.2
11.9 10.7 8.6 11.6 9.6 11.8 11.5 10.7 8.6 11.1 10.6 þ/ 1.3
Values are the mean of determinations made in triplicate. UA ¼ uronic acid. A:X ¼ ratio ara:xyl. a BSG from ale-producing breweries b BSG from lager-producing brewery
204.2 181.7 203.1 197.4 238.3 206.1 137.0 181.1 173.4 174.2 189.7 þ/ 26.9
54.7 46.7 31.7 47.2 47.9 38.5 50.9 51.0 25.9 47.3 44.2 þ/ 9.2
A:X SD
544.1 0.8 485.8 86.8 476.0 43.3 511.7 13.3 530.7 10.7 557.0 7.9 445.3 9.4 480.4 15.1 459.3 18.8 488.6 19.7 497.9 þ/ 36.8
0.41 0.48 0.49 0.48 0.54 0.46 0.55 0.52 0.44 0.54 0.50 þ/ 0.05
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Table 7 Component alkali-extractable phenolic acids determined in AIR preparations from BSG (mg g1 AIR). Sample code a
P01 001 P01 002a P01 003a P01 004a P08 001a P02 001b P05 001b P07 001b P09 001b P11 001b Mean þ/ SD a b
Coumaric Acid
8,80 ; 6,70 -DiFA
Ferulic acid
8,80 -DiFA
8,50 -DiFA
5,50 -DiFA
8-0-40 -DiFA
8,50 ; 7-O-40 -DiFA
Total þ/ SD
3.2 2.0 1.4 2.1 1.7 2.0 1.8 1.3 1.5 1.4 1.8 þ/ 0.6
0.4 0.8 0.8 0.1 0.6 0.6 0.9 0.5 1.0 0.9 0.7 þ/ 0.3
3.9 6.3 4.4 6.7 5.2 3.8 6.0 3.9 3.9 5.3 4.9 þ/ 3.0
0.0 0.1 0.1 0.5 0.1 0.1 0.1 0.1 0.1 0.4 0.2 þ/ 0.2
0.2 0.4 0.5 0.5 0.2 0.4 0.0 0.4 0.6 0.0 0.3 þ/ 0.2
0.5 0.9 0.5 0.9 0.7 0.6 0.9 0.5 0.5 0.9 0.7 þ/ 0.2
1.6 1.7 1.2 2.0 1.4 1.5 1.4 1.3 1.3 1.3 1.5 þ/ 0.2
0.0 0.0 0.1 0.0 0.1 0.3 0.0 0.2 0.1 0.2 0.1 þ/ 0.1
10.22.0 12.42.6 9.40.7 12.91.7 10.20.3 9.40.3 11.53.3 8.40.4 9.11.2 10.60.8 10.4 þ/ 1.5
BSG from ale-producing breweries BSG from lager-producing brewery. DiFA ¼ diferulic acid
the difference in BSG is readily distinguished by beer type produced then this can be taken into account when selecting BSG for downstream processing. 3.2.2. Polysaccharides Polysaccharides, as NSP and starch, accounted for approximately 50% of the AIR in BSG. From the predominance of arabinose, xylose and glucose (Table 5) the major polysaccharides were inferred to be arabinoxylan (AX) and glucan. The ara:xyl ratio for all samples (0.50 þ/ 0.05) was similar and similar to previously reported ratios (Mandalari et al., 2005), suggesting that malt source has little effect on AX composition in BSG. The low content of galactose (Table 5) indicates that arabinogalactans (AG) (Loosveld, Grobet, & Delcour, 1997) are a minor component of the BSG. Cellulose, estimated as the difference between total glucan and polymeric starch was 19.7% þ/ 2.7 of the original BSG dry weight. This estimate also includes a contribution from the low levels of mixed-linkage present in the glucan, (w1%: see below), and compares with a reported cellulose content of BSG w17%, (Mussatto et al., 2006; Valverde, 1994). Detailed analysis for total starch-based material and mixedlinkage b-glucans (Table 6) indeed showed BSG to contain a variable but significant amount of residual starch (16–130 mg g1). BSG from lager producers contained 35.8 mg g1 starch whilst from ale producers this was 97.6 mg g1 starch. Whilst overlap exists in the starch content of BSG from breweries producing either ale or lager, an analysis of variance (Table 4) showed there was a significant
difference (p < 0.001) between beer types. Starch analysis on the AIR compared to the BSG prior to AIR preparation showed that a proportion of the total starch could be accounted for as lower molecular weight (alcohol soluble) maltodextrins. These maltodextrins represented approximately 37% of the total starch measured and this proportion was similar for all breweries. At a ratio of w60% amylose in the residual starch, this amylose content was higher than expected for barley starch (w25% proportion of amylose) (Tester, 1997; You & Izydorczyk, 2002). Although this suggests there may be some resistance to digestion of amylose during mashing, the levels of residual starch indicate there is little apparent effect on the overall digestion of starch either between malt types or between breweries. Mixed linkage b-glucan in BSG, 7 mg g1 dry weight, was similar for all breweries. The much reduced content of mixed-linkage b-glucan in BSG compared to barley grain (25–54 mg g1) and malt (Manzanares & Sendra, 1996; Papageorgiou, Lakhdara, Lazaridou, Biliaderis, and Izydorczyk (2005); Gamlath, Aldreda, & Panozzo, 2008) demonstrates an extensive degradation of mixed-linkage b-glucan during mashing and hence that BSG is unsuitable for exploitation as a source of mixed-linkage b-glucan in functional foods. 3.2.3. Cell wall-bound phenolics Bound phenolics are a minor component in BSG AIR (Table 7) and predominantly based on ferulic acid and its dehydrodimers. The spectrum and concentration of bound phenolics is also similar
tryptophan arginine lysine histidine phenylalanine tyrosine leucine isoleucine valine glycine alanine proline glutamic acid / glutamine aspartic acid / asparagine serine threonine hydroxproline methionine cysteine 0
5
10
15
20
25
Amino Acid (g/100g amino acid) Fig. 1. Profile of amino acid determined in BSG samples. Samples, in triplicate, corresponding to 10 mg nitrogen (N) were hydrolysed with 6 M HCl for 23 hours at pH 2.2. Hydrolysates were analysed on a Biochrom 30 Amino Acid Analyser, except for tryptophan, which was analysed by HPLC. (black) ¼ ale; (grey) ¼ lager. The values for ale correspond to samples 1 – 5 (Table 3) and for lager to samples 6 – 10 (Table 3). Values are shown as the mean þ 1sd.
J.A. Robertson et al. / LWT - Food Science and Technology 43 (2010) 890–896
for all samples. On the basis of the overall arabinose content and ferulate-based components in BSG then an estimated 1 in 12 arabinose units is esterified to a ferulate. Also, around 60% of the esterified ferulate is estimated to be present as ferulic acid, with the remaining 40% involved in dehydrodimer crosslinking. When crosslinking is expressed as the ratio of xylose to dehydrodimers (Antoine et al., 2003), the gross index of cross linking for BSG is w52. This shows BSG to be a highly crosslinked material, much more so than wheat bran (Antoine et al., 2003). Overcoming the crosslinking to control the release of component polysaccharides and protein is seen as a major challenge to the exploitation of BSG (Robertson, Faulds, Smith, & Waldron, 2008). 3.2.4. Protein and amino acids Variation in protein content of BSG between breweries (Table 4) was ascribed to the malt/beer source, with BSG from lager production having a significantly higher protein content (223.1 mg g1 þ/ 14.2) compared to BSG from ale production (149.2 mg g1 þ/ 32.1). However, amino acid analysis (Fig. 1) gave a similar profile of amino acids for ale and lager sourced BSG. The predominant amino acid was glutamine/glutamic acid, as expected since the glutamine-rich barley storage proteins, hordeins, are a major component of the protein present in BSG. During mashing, hordeins are involved in extensive disulphide bond formation, resulting in an aggregate formation and reduction in solubility (Celus et al., 2006). Aggregate formation would account for the persistence of hordeins in AIR preparations and their presence may also form a constraint on the solubilisation of components within the BSG matrix; as suggested by a recent study showing that disruption of the protein matrix by peptidases enhances the effectiveness of glycoside hydrolases and esterase on BSG (Faulds et al., 2009). On the other hand, it remains fully possible to solubilize most of the protein in BSG using a mild alkaline extraction and/or enzymatic methods (Celus, Brijs, & Delcour, 2007; Treimo, Aspmo, Eijsink, & Horn, 2008). 4. Conclusion The analysis of fresh samples of BSG obtained on the day of production, from a range of breweries, has shown that BSG from lager brewing has a higher protein content but lower residual starch content than BSG from ale brewing. These differences are consistent with the different source malts. In general, the nonstarch polysaccharide profile of BSG is a relatively uniform between breweries, as is the protein composition. Lignin content and cell wall bound phenolics also show little variation between breweries. Thus, as a feedstock available for industrial upgrading differences between beer type brewed is the major source variation. Since differences in protein and starch content can be identified from the type of beer being produced this should not present problems with a routine industrial upgrading of BSG. However, the high moisture content and composition of BSG makes it highly susceptible to microbial degradation. It is the presence of a resident microflora which is most likely to affect the potential to use BSG as a reliable and uniform industrial feedstock for value-added downstream processing. However, at the point of production, microbial contamination is low and considered within acceptable limits for food use, but appropriate storage regimens will be required to minimise microbial proliferation prior to downstream processing of the BSG. Acknowledgements We gratefully acknowledge funding from the Biotechnology and Biological Sciences Research Council (BBSRC) UK, and the
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