International Journal of Food Microbiology 173 (2014) 36–40
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short communication
Malting of barley with combinations of Lactobacillus plantarum, Aspergillus niger, Trichoderma reesei, Rhizopus oligosporus and Geotrichum candidum to enhance malt quality M. Hattingh a, A. Alexander b, I. Meijering b, C.A. van Reenen a, L.M.T. Dicks a,⁎ a b
Department of Microbiology, University of Stellenbosch, Stellenbosch 7600, South Africa Southern Associated Maltsters (Pty) LTD, P.O. Box 27, Caledon 7230, South Africa
a r t i c l e
i n f o
Article history: Received 14 August 2013 Received in revised form 10 December 2013 Accepted 20 December 2013 Available online 28 December 2013 Keywords: Malting Lactobacillus plantarum Aspergillus Trichoderma Rhizopus Geotrichum
a b s t r a c t Good quality malt is characterised by the presence of high levels of fermentable sugars, amino acids and vitamins. To reach the starch-rich endosperm of the kernel, β-glucan- and arabinoxylan-rich cell walls have to be degraded. β-Glucanase is synthesized in vast quantities by the aleurone layer and scutellum during germination. Secretion of hydrolytic enzymes is often stimulated by addition of the plant hormone gibberellic acid (GA3) during germination. We have shown an enhanced β-glucanase and α-amylase activity in malt when germinating barley was inoculated with a combination of Lactobacillus plantarum B.S1.6 and spores of Aspergillus niger MH1, Rhizopus oligosporus MH2 or Trichoderma reesei MH3, and L. plantarum B.S1.6 combined with cell-free culture supernatants from each of these fungi. Highest malt β-glucanase activity (414 Units/kg malt) was recorded with a combination of L. plantarum B.S1.6 and spores of A. niger MH1. Highest α-amylase activities were recorded with a combination of L. plantarum B.S1.6 and spores of R. oligosporus MH2 (373 Ceralpha Units/g malt). Highest FAN levels were recorded when L. plantarum was inoculated in combination with spores of either R. oligosporus MH2 or T. reesei MH3 (259 and 260 ppm, respectively). This is the first study showing that cell-free culture supernatants of Aspergillus, Rhizopus and Trichoderma have a stimulating effect on β-glucanase and α-amylase production during malting. A combination of L. plantarum B.S1.6, and spores of A. niger MH1 and R. oligosporus MH2 may be used as starter cultures to enhance malt quality. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Barley malt is the predominant raw material used in beer production (Haraldsson et al., 2004) and the quality thereof is characterised by the presence of high levels of fermentable sugars, amino acids and vitamins (Justé et al., 2011; Kreisz, 2009; Laitila et al., 2006a). During germination of barley, β-glucan is degraded by β-glucanase, synthesized in vast quantities by the aleurone layer and scutellum (Hrmova and Fincher, 2001). This leads to the production of (1,3;1,4)-β-D-triand tetrasaccharides, although oligosaccharides of up to 10 units may also form. Slow, or inadequately degraded β-glucan results in a high polymer content that leads to a hazy malt (Jin et al., 2004), high viscosity and poor lautering performance (Ullrich, 2011). To enhance the malting process, brewers often add gibberellic acid (GA3) to barley to break dormancy and reduce germination time (Kreisz, 2009; Noots et al., 1999). The addition of GA3, however, has to be carefully controlled. Excessive levels facilitate extensive rootlet formation and may lead to abnormally high sugar and soluble nitrogen levels, which in turn leads to abnormal colour development (Fox et al., ⁎ Corresponding author. Tel.: +27 21 808 5849. E-mail address: [email protected] (L.M.T. Dicks). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.12.017
2003). This led scientists searching for alternative methods to enhance germination (Goode and Arendt, 2006). Acidification of barley mash with Lactobacillus amylovorus improved fermentability and FAN content, and reduced β-glucan levels in wort (Lowe et al., 2004, 2005). This was attributed to the lowering of pH and the production of proteolytic and amylolytic enzymes. Laitila et al. (2006a) reported a reduction in wort viscosity and β-glucan content, and also an increase in xylanase and β-glucanase activities when mash was inoculated with Lactobacillus plantarum and Pediococcus pentosaceus. In other studies, where combinations of lactic acid bacteria and Geotrichum candidum were used, malt quality improved by restricting the growth of unwanted contaminants (Biovin and Malanda, 1997; Laitila et al., 2006b; Linko et al., 1998; Lowe et al., 2005). In one study a Rhizopus oligosporus starter culture enhanced malting (Noots et al., 2001). Apart from the possible production of proteolytic and amylolytic enzymes, lactic acid bacteria are known to produce antimicrobial peptides (bacteriocins) and antifungal compounds that may restrict the growth of bacteria and fungi. From these and similar studies (Foszcynska et al., 2004; Haikara et al., 1993; Noots et al., 1999), it is clear that the development of starter cultures containing lactic acid bacteria, separately or combined with fungi, may be an attractive alternative to chemical and enzymatic treatment of mash.
M. Hattingh et al. / International Journal of Food Microbiology 173 (2014) 36–40
The aim of this study was to determine whether strains of L. plantarum and spores of Aspergillus niger, R. oligosporus or Trichoderma reesei, originally isolated from barley, and G. candidum isolated from barley malt, could be used as starter cultures to enhance the malting process. To evaluate the performance of these strains during malting, the activity of α-amylase, β-glucanase, xylanase, cellulose and proteases, and the level of free amino nitrogen (FAN) content, was tested. 2. Material and methods 2.1. Microbial strains L. plantarum B.S1.6, A. niger MH1, T. reesei MH3 and R. oligosporus MH2 were originally isolated from barley. L. plantarum B.S1.6 was cultured in De Man, Rogosa and Sharpe (MRS) broth (Biolab, Biolab Diagnostics, Midrand, South Africa) at 30 °C, and strains MH1, MH3 and MH2 in Yeast Peptone Dextrose (YPD) medium (Biolab) at 26 °C. Geotrichum strains, isolated from barley malt, were identified as G. candidum based on sequencing of the D1/D2 region of the 26S rDNA (of rRNA gene), amplified with forward primer F63 (5′-GCATATCAAT AAGCGGAGGAAAAG-3′) and reverse primer LR3 (5′-GGTCCGTGTTTC AAGACGG-3′). The method of Garner et al. (2010) was used.
37
Actively growing cells of A. niger MH1, T. reesei MH3, R. oligosporus MH2 and the 12 strains of G. candidum were inoculated into Erlenmeyer flasks with 50 mL SC-medium, supplemented with either 1.0% (w/v) low viscosity CMC, 0.1% (w/v) β-glucan, 0.5% (w/v) beechwood xylan or 2.0% (w/v) starch. Incubation was on a rotary shaker (250 rpm) at 26 °C for 7 days. The cells were harvested (10,000 ×g, 15 min, 4 °C) and enzyme activities determined in the cfs. Cellulase and β-glucanase activities were determined by adding 20 μL 0.1% (w/v) CMC and 0.1% (w/v) β-glucan, each suspended in 0.05 M citrate buffer (pH 4.8), to 30 μL cfs in respective wells of a microtitre plate. The microtitre plate was incubated at 50 °C for 30 min and enzymatic reactions stopped by the addition of 100 μL DNS solution (1.0%, v/v, 3,5-dinitrosalicyclic acid; 20.0%, w/v, potassium sodium tartrate; 1.0%, w/v, NaOH; 0.2%, v/v, phenol and 0.05%, w/v, Na2SO3), followed by boiling for 15 min. The samples were allowed to cool to 25 °C and readings were taken at 540 nm (Bio Rad, Smartspec Plus). The same protocol was used to determine xylanase activity, except that 6 μL cfs was added to 54 μL of 1.0% (w/v) beechwood xylan, suspended in 0.05 M citrate buffer, pH 4.8 (Miller et al., 1960). The samples were incubated for 60 min and the reaction stopped by the addition of 90 μL DNS solution. Activity was expressed in nKats/mL. α-Amylase was determined using the Ceralpha method and activity expressed as CU/mL. The G. candidum strain with the highest overall enzyme activities was selected for further studies.
2.2. Enzyme activity assays 2.3. Preparation of starter cultures 2.2.1. Screening of cultures for the production of enzyme activity L. plantarum B.S1.6 was cultured in MRS broth at 30 °C for 8 h. Five microlitres of the culture was used to inoculate the following: MRS agar, supplemented with 1.4% (w/v) skimmed milk powder (Difco, Becton, Dickinson and Company, Sparks, Maryland, USA), 1.0% (w/v) carboxymethylcellulose (CMC) (Sigma-Aldrich), 0.1% (w/v) β-glucan (Difco), 0.5% (w/v) beechwood xylan (Carl Roth GmbH, Karlsruhe, Germany) or 2% (w/v) soluble starch (Merck, Darmstadt, Germany). After 48 h of incubation at 30 °C, substrate hydrolysis was detected as clear zones by positive colonies through flooding the plates with appropriate dyes. Plates containing CMC, β-glucan and xylan as substrates were stained with 0.1% (w/v) Congo red (Sigma-Aldrich) for 15 min. Excess dye was rinsed off with 1 M NaCl. Enzyme activity was enhanced by destaining plates with 1 M HCL, as described by Ruijssenaars and Hartmans (2001). Starch hydrolysis was observed by flooding the plates with iodine, while proteolytic zones were enhanced by adding 10% (v/v) tannic acid. Protease, cellulase, β-glucanase, xylanase and α-amylase activities of A. niger MH1, T. reesei MH3, R. oligosporus MH2 and the 12 Geotrichum strains were tested using the following protocol. Protease activity was determined on 1.4% (w/v) skimmed milk agar, cellulase activity on 0.67% (w/v) YNB agar (Sigma-Aldrich) supplemented with 1.0% (w/v) CMC, β-glucanase activity on YPD agar supplemented with 0.1% (w/v) β-glucan, as described by Strauss et al. (2001), xylanase activity on Potato Dextrose agar (PDA, Biolab) supplemented with 0.5% (w/v) beechwood xylan, and α-amylase on Nutrient agar (Biolab) and YNB agar (0.67%), each supplemented with 2.0% (w/v) soluble starch, as described by Buzzini and Martini (2002). The plates were incubated at 26 °C for 7 days and then tested for enzyme activity as described before. 2.2.2. Quantification of enzyme activities Enzyme reactions recorded with plate assays were quantified using spectrophotometric methods. Actively growing cells of L. plantarum B.S1.6 were inoculated into 50 mL MRS broth, supplemented with 2% (w/v) starch. After 40 h of incubation at 30 °C, the cells were harvested (10 000 ×g, 15 min, 4 °C) and 30 μL of each cell-free culture supernatant (cfs) added to wells in a 96-well microtitre plate. The plate was pre-incubated for 5 min at 50 °C. α-Amylase was determined using the Ceralpha method (Megazyme, International Ireland, Ltd, County Wicklow, Ireland) and activity expressed as Ceralpha units (CU) per mL.
L. plantarum B.S1.6 was cultured in MRS broth at 30 °C for 48 h, the cells harvested (10,000 ×g, 10 min, 10 °C) and resuspended in sterile distilled water to yield 8 × 106 cfu/20 mL. R. oligosporus MH2 and A. niger MH1 were streaked onto PDA and T. reesei MH3 onto Malt Extract Agar (MEA, Biolab), and incubated at 26 °C for 7 days. G. candidum strain 1173 was streaked onto YPD agar and incubated at 26 °C for 48 h. Spores of Rhizopus sp. MH2, T. reesei MH3, A. niger MH1 and G. candidum 1173 were washed from plates with 5 mL sterile physiological saline, counted using a haemocytometer (Paul Marienfeld GmbH & Co, Germany) and 8 × 108 spores suspended into 20 mL sterile distilled water. 2.4. Preparation of cell-free supernatant R. oligosporus MH2, T. reesei MH3 and A. niger MH1 were each cultured in 100 mL YNB, supplemented with 1.0% (w/v) low viscosity CMC, 0.1% β-glucan, 0.5% (w/v) beechwood xylan and 2.0% (w/v) starch, respectively. All cultures were incubated aerobically, on a rotary shaker (250 rpm) at 26 °C for 7 days. Cell-free culture supernatants were collected separately by filtration through sterile 0.22 μm nitrocellulose membranes. Cellulase, β-glucanase and xylanase activities were determined using the DNS-assay and α-amylase activity using the Ceralpha method, as described elsewhere. Twenty microlitres of the cfss of A. niger MH1, T. reesei MH3 and Rhizopus sp. MH2 was used to inoculate the barley. 2.5. Malting Eight micro-malting trials were conducted in separate 5 L plastic containers (strains MH1 + B.S1.6, MH2 + B.S1.6, MH3 + B.S1.6, 1173 + B.S1.6, MH1 cfs + B.S1.6, MH2 cfs + B.S1.6, MH3 cfs + B.S1.6 and the control, not inoculated with cells or cfs). Barley (500 g, Erica cultivar) was added to a 5 L plastic container with holes (1 mm in diameter) drilled into the bottom of the container. The container was placed inside an empty 5 L container linked to an air pump. Sterile distilled water (800 mL) was added to the barley in the inner container. The barley kernels were steeped for 43 h (9 h wet stand, followed by 14 h air rest, 14 h wet stand and 6 h air rest), germinated for 84 h and then kilned in five stages (the length of each stage: 3, 5, 12, 4 and 2 h). At the start of germination, the barley kernels were inoculated
38
M. Hattingh et al. / International Journal of Food Microbiology 173 (2014) 36–40
Table 1 Hydrolysis of casein, cellulose, β-glucan, xylan and starch (enzyme activities are shown in parenthesis). Strain
Hydrolysis of:
L. plantarum B.S1.6 A. niger MH2 T. reesei MH3 Rhizopus oligosporus MH2 G. candidum 1173 a b
Casein
Cellulosea
β-Glucana
Xylana
Starchb
− − − − +
− (4.7 ± 0.4) (17.6 ± 2.7) (0.8 ± 0.1) (0.5 ± 0.3)
− (7.4 ± 0.7) (10.9 ± 0.6) (0.6 ± 0.1) (0.4 ± 0.1)
− (217.6 ± 12.7) (53.9 ± 4.3) (0.6 ± 0.2) (0.9 ± 0.2)
+ (33.7 ± 5.2) (171.0 ± 8.11) −
Enzyme activity in nkats/mL. Amylase activity in CU/mL.
with 20 mL L. plantarum B.S1.6 (8 × 106 cfu) and 20 mL (8 × 108 spores) of either A. niger MH1, T. reesei MH3, R. oligosporus MH2 or G. candidum 1173. This corresponded to 1.6 × 104 cfu L. plantarum B.S1.6 and 1.6 × 106 spores per gramme barley. After germination, samples were collected and viable cell numbers of L. plantarum B.S1.6 determined by plating onto MRS agar. Cell numbers of A. niger MH1, T. reesei MH3 and R. oligosporus MH2 were determined by plating onto MEA, and cell numbers of G. candidum 1173 by plating onto YPD. All plates were incubated for 48 h at 26 °C. In another experiment, 20 mL cfs of either strain MH1, MH3 or MH2 were added to 20 mL L. plantarum B.S1.6. The control received 20 mL sterile distilled water. Germination was for 84 h, followed by kilning for 26 h. 2.6. Malt analyses Kilned malt was analysed according to methods used by the European Brewing Convention (Bamforth and Barclay, 1993). In brief, this entailed grinding of 50 g kilned malt in a disc mill (Bühler-Miag 4000, Bühler Ltd., Milan, Italy) with pore sizes of 0.2 mm. Distilled water (400 mL) was added to the grinded malt and the suspension placed in a mash bath (Industrial Equipment Corporation). The mash was filtered and the β-glucan and free-amino nitrogen (FAN) content in the wort determined according to methods used by the American Society of Brewing Chemists (2009). Beta-glucanase activity in the malt was determined using the Azo-barley glucan method (K-MBGL 03/11, Megazyme). Reactions were conducted at 30 °C and 60 °C to differentiate between malt β-glucanase and microbial β-glucanase activity, respectively (Home et al., 1993; Laitila et al., 2006a). Xylanase activity was determined using the Xylazyme AX method (T-XAX200 10/2008, Megazyme). Xylanase enzyme extraction was performed for 15 min at room temperature in sodium acetate buffer (25 mM, pH 4.7), with continuous stirring. α-Amylase activity was quantified with the Ceralpha kit (K-CERA 08/05, Megazyme), as described before. 2.7. Statistical analyses Statistical analyses were done using Statistica v. 10 (StatSoft, Inc.). The students' T-test was performed at 95% confidence levels. Significant differences (p = 0.05) were indicated by an asterisk (*).
3. Results and discussion 3.1. Enzyme production and activities Hydrolysis of casein, cellulose, β-glucan, xylan and starch, and activity of α-amylase is listed in Table 1. The hydrolysis of starch and α-amylase by L. plantarum is characteristic of the species (Hammes and Hertel, 2009). Most lactic acid bacteria, including L. plantarum, do not have proteolytic, cellulase, β-glucanase or xylanase activities (Hammes and Hertel, 2009; Okano et al., 2010). However, a few exceptions have been reported. Khalid and Marth (1990) described the strains of L. plantarum with the ability to hydrolyse casein, and Kilic and Akpinar (2013) have shown that low concentrations of β-glucan (0.5% and 1.0%, w/v) enhanced the growth of probiotic strains of L. plantarum. Attempts to genetically engineer strains of L. plantarum to degrade xylan, cellulose and β-glucan have been successful (Moraïs et al., 2013; Rossi et al., 2001). A. niger MH1, T. reesei MH3 and R. oligosporus MH2 did not hydrolyse casein on skimmed milk plates and no proteolytic activity was detected in the cfs (Table 1). Not all strains of Aspergillus, Trichoderma and Rhizopus hydrolyse casein, as shown by Sohail et al. (2009). Of the nine proteolytic G. candidum isolates, strain 1173 formed the largest zone of casein hydrolysis. Casein hydrolysis is a well-documented characteristic of G. candidum (Ahearn et al., 1968). A. niger MH1, T. reesei MH3, R. oligosporus MH2 and all 12 strains of G. candidum hydrolysed cellulose and β-glucan on plates and would thus be able to degrade the cell walls protecting the starch-rich grains in the endosperm. A. niger MH1, T. reesei MH3, R. oligosporus MH2, and three of the 12 G. candidum isolates, including strain 1173, hydrolysed xylan (Table 1). Degradation of xylan is common amongst fungi (Sohail et al., 2009). A. niger MH1 and R. oligosporus MH2 hydrolysed starch on plates and produced α-amylase at 33.66 ± 5.23 and 171 ± 8.11 CU/mL, respectively, in cfs (Table 1). Amylase production by Aspergillus and Rhizopus spp. is well reported (Sohail et al., 2009). Starch hydrolysis by Geotrichum spp., on the other hand, is less common and only a few strains with amylase activity have been reported. Arotupin (2007) isolated an amylase-positive strain of G. candidum from Cassava waste water and Falih (1998) described two amylase-producing G. candidum strains isolated from soil. Of the 10 Geotrichum klebahnii strains described by Buzzini and Martini (2002), only one produced an amylase.
Table 2 Enzyme activities in malt produced with L. plantarum B.S1.6, in combination with viable cells and cell-free supernatant (cfs) of A. niger MH1, R. oligosporus MH2, T. reesei MH3 and G. candidum. Significantly increased enzyme activities are indicated by*. Malting trial
α-Amylase (CU/g malt)
β-Glucanase (U/kg malt at 30 °C)
β-Glucanase (U/kg malt at 60 °C)
Total β-glucanase (U/kg at 30 °C + 60 °C)
Xylanase (U/g malt)
A. niger MH1 R. oligosporus MH2 T. reesei MH3 G. candidum 1173 niger MH1 cfs R. oligosporus MH2 cfs T. reesei MH3 cfs Control
355* 373* 338* 166 314* 365* 346* 151
414* 282* 347* 207 324* 343* 293 155
274* 203* 228* 131 201* 243* 189* 78
688* 485* 575* 338 525* 586* 482* 232
0.29 0.33 0.30 0.25 0.39* 0.46* 0.37* 0.22
M. Hattingh et al. / International Journal of Food Microbiology 173 (2014) 36–40
L. plantarum B.S1.6 and spores of T. reesei MH3 decreased the β-glucan in malt from 60 ppm to 54 and 49 ppm, respectively (Fig. 1). The greater decrease in β-glucan content in the presence of L. plantarum B.S1.6 and T. reesei MH3 co-insides with the high β-glucanase activity recorded in the cfs of strain MH3 (10.85 ± 0.56). Less β-glucan was degraded when barley was inoculated with a combination of L. plantarum B.S1.6 and spores of R. oligosporus MH2, as indicated by a decrease from 60 ppm to 57 ppm (Fig. 1). A similar poor degradation of β-glucan (from 60 ppm to 59 ppm) was recorded when barley was inoculated with a combination of L. plantarum B.S1.6 and G. candidum 1173 (Fig. 1). This corresponds with the weak β-glucanase activities recorded in the cfs of these strains. Barley inoculated with L. plantarum B.S1.6 and spores of A. niger MH1, or L. plantarum B.S1.6 and spores of T. reesei MH3 had the highest total β-glucanase activities when measured at 30 °C and 60 °C (688 and 575 U/kg malt, respectively; Table 2). The overall β-glucanase activity recorded when barley was inoculated with L. plantarum B.S1.6 and spores of Rhizopus sp. MH2 was much lower (485 U/kg malt, Table 2). Aspergillus spp. and Rhizopus spp. are used as starter cultures in Asian fermented cereals. Rhizopus spp. has been used to enhance malt modification (Biovin and Malanda, 1997). L. plantarum B.S1.6 was selected based on its ability to produce cell-bound and extracellular α-amylase (Hattingh, 2013). The total β-glucanase activities (malt and microbial enzyme activities) produced during malting by starter cultures containing spores of A. niger MH1 or T. reesei MH3 were higher than β-glucanase activities produced in the presence of cfss of Aspergillus sp. MH1 and T. reesei MH3 (Table 2). However, much higher β-glucanase activities were recorded in malt inoculated with cfs of R. oligosporus MH2 (586 U/kg malt) compared to spores (485 U/kg malt, Table 2). The FAN content reflects the extent at which proteolysis takes place during malting and mashing. A high FAN content is essential to ensure active fermentation by the brewers' yeast (Lowe et al., 2004). The FAN content of kilned malt prepared without starter cultures or cfs was 205 ppm and much lower compared to the FAN content recorded when barley was inoculated with starter cultures or cfss (Fig. 2). Highest FAN levels were recorded when L. plantarum was inoculated in combination with spores of either Rhizopus sp. MH2 or T. reesei MH3 (259 and 260 ppm, respectively; Fig. 2). Slightly higher FAN levels were recorded when cfs of A. niger MH1 or R. oligosporus MH2 (267 ppm; Fig. 2) were used. The latter combination is thus preferred. Highest increase in α-amylase activity (373 CU/g malt) was recorded when malting was performed in the presence of L. plantarum B.S1.6 and
70 60 50 *
* *
40 30 20 10 0 Aspergillus Rhizopus sp. sp. MH1 MH2
T. reesei MH3
G. candidum Aspergillus Rhizopus sp. 1173 sp. MH1 cfs MH2 cfs
T. reesei MH3 cfs
39
Control
Malting trial Fig. 1. Beta-glucan content of kilned malt from seven malting trials and the control. Lactobacillus plantarum B.S1.6 was combined with the respective starter cultures. Significant changes in β-glucan content are indicated by *.
None of the Geotrichum isolates studied by Laitila et al. (2006b), Noots et al. (2001) and Subash et al. (2005) degraded starch. Of the 12 G. candidum isolates tested, strain 1173 produced the highest level of cellulose, β-glucanase and xylanase activities and was selected as starter culture. 3.2. Malting The cell numbers of L. plantarum B.S1.6 decreased from 1.6 × 104 cfu to 1 × 104 cfu per gramme malt and the spore count of A. niger MH1, T. reesei MH3 and R. oligosporus MH2 from 1.6 × 106 cfu to 1.0 × 104 cfu per gramme malt after 84 h of germination. The cell numbers of G. candidum 1173 decreased from 1.6 × 106 cfu to 1.0 × 104 cfu during the same period. The total β-glucanase activity in the control malt, i.e. β-glucanase recorded for malt (readings taken at 30 °C) plus β-glucanase related to microbial activity (readings taken at 60 °C), was 233 U/kg malt (Table 2). In all experiments with control malt the β-glucanase activity pertaining to microbial activity was always lower, suggesting that microorganisms naturally present in barley contribute very little to the production of β-glucanase. Inoculation of barley with a combination of L. plantarum B.S1.6 and spores of A. niger MH1, or
* 280 260
*
*
* *
240
FAN (ppm)
*
*
220 200 180 160 140 120 100 Aspergillus Rhizopus sp. MH1 sp. MH2
T. reesei MH3
G. Aspergillus Rhizopus T. reesei candidum sp. MH1 cfs sp. MH2 cfs MH3 cfs 1173
Control
Malting trial Fig. 2. Free amino nitrogen (FAN) content of kilned malt recorded for seven malting trials and the control. Lactobacillus plantarum B.S1.6 was combined with the respective starter cultures. Significant changes in FAN content are indicated by *.
40
M. Hattingh et al. / International Journal of Food Microbiology 173 (2014) 36–40
spores of R. oligosporus MH2 (Table 2). Xylanase activities were very low and compared to that recorded in the control malting trial (Table 2). 4. Conclusion Concluded from the seven malting trials, β-glucan was most actively degraded in the presence of L. plantarum B.S1.6 and cfs of A. niger MH1, Rhizopus sp. MH2 and T. reesei MH3. Highest FAN levels were recorded when L. plantarum was inoculated in combination with spores of either R. oligosporus MH2 or T. reesei MH3. Malting could be enhanced by inoculating barley with a combination of L. plantarum B.S1.6, A. niger MH1, R. oligosporus MH2 and T. reesei MH3. Large scale brewing experiments will have to be conducted to evaluate the starter cultures. Acknowledgements The National Research Foundation (NRF) of South Africa for a bursary to M. Hattingh is acknowledged. References Ahearn, D.G., Meyers, S.P., Nichols, R.A., 1968. Extracellular proteinases of yeasts and yeastlike fungi. Appl. Microbiol. 16, 1370–1374. American Society of Brewing Chemists, 2009. Methods of analysis, Statistical Analysis-2 Limits of Detection and Determination2009 edition. The Society, St. Paul, MN. Arotupin, D.J., 2007. Evaluation of microorganisms from cassava waste water for production of amylase and cellulase. Res. J. Microbiol. 2, 475–480. Bamforth, C.W., Barclay, A.H.P., 1993. Malting technology and the use of malt. In: MacGregor, A.W., Bhatty, R.S. (Eds.), Barley: Chemistry and Technology. American Association of Cereal Chemists, St. Paul, Minnesota. Biovin, P., Malanda, M., 1997. Improvement of malt quality and safety by adding starter culture during the malting process. Master Brew. Assoc. Am. 34, 96–101. Buzzini, P., Martini, A., 2002. Extracellular enzyme activity profiles in yeast and yeast-like strains isolated from tropical environments. J. Appl. Microbiol. 93, 1020–1025. Falih, A.M., 1998. Effect of heavy-metals on amylolytic activity of the soil yeasts Geotrichum captatum and Geotrichum candidum. Bioresour. Technol. 66, 213–217. Foszcynska, B., Dzubia, E., Stempniewicz, R., 2004. The use of Geotrichum candidum starter culture for protection of barley and its influence on biotechnological qualities of malts. Electron. J. Pol. Agric. Univ. 7. Fox, G.P., Panozzo, J.F., Li, C.D., Lance, R.C.M., Inkerman, P.A., Henry, R.J., 2003. Molecular basis of barley qualities. Aust. J. Agric. Res. 54, 1081–1101. Garner, C.D., Starr, J.K., McDough, P.L., Altier, C., 2010. Molecular identification of veterinary yeast isolates by use of sequence-based analysis of the D1/D2 region of the large ribosomal subunit. J. Clin. Microbiol. 48, 2140–2146. Goode, D.L., Arendt, E.K., 2006. Development in the supply of adjunct materials for brewing. In: Bamforth, C.W. (Ed.), Brewing, New Technologies. Woodhead Publishing Ltd, Cambridge, England, pp. 30–58. Haikara, A., Uljas, H., Suurnaki, A., 1993. Lactic acid starter cultures in malting — a novel solution to gushing problems. Proceedings of the European Brewery Convention Congress, Lisbon, 24. IRL Press, Oxford, pp. 163–172. Hammes, W.P., Hertel, C., 2009. Genus I. Lactobacillus Beijerinck 1901, 212AL, In: De Vos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A., Schleifer, K.-H., Whitman, W.B. (Eds.), Bergey's manual of systematic bacteriology, 2nd edition. The Firmicutes, vol. 3. Springer, New York, pp. 465–511. Haraldsson, A., Rimsten, L., Alminger, M.L., Andersson, R., Andid, T., Aman, P., Sandberg, A., 2004. Phytate content is reduced and β-glucanase activity suppressed in malted barley steeped with lactic acid at high temperature. J. Sci. Food Agric. 84, 653–662.
Hattingh, M., 2013. The Effect of Lactic Acid Bacteria and Fungi on the Malting of Barley. (MSc thesis) University of Stellenbosch, Stellenbosch, South Africa. Home, S., Pietila, K., Sjöholm, K., 1993. Control of glucanolysis on mashing. J. Am. Soc. Brew. Chem. 51, 108–113. Hrmova, M., Fincher, G.B., 2001. Structure-function relationships of β-D-glucan endo- and exohydrolyses from higher plants. Plant Mol. Biol. 47, 73–91. Jin, Y.L., Speers, R.A., Paulson, A.T., Stewart, R.J., 2004. Barley β-glucans and their degredation during malting and brewing. Master Brew. Assoc. Am. Tech. Q. Pap. 41, 231–240. Justé, A., Malfliet, S., Lenaerts, M., de Cooman, L., Aerts, G., Willems, K.A., Lievens, B., 2011. Microflora during malting of barley: overview and impact on malt quality. Brew. Sci. 64, 22. Khalid, N., Marth, E.H., 1990. Proteolytic activity by strains of Lactobacillus plantarum and Lactobacillus casei. J. Dairy Sci. 73, 3068–3076. Kilic, G.B., Akpinar, D., 2013. The effects of different levels of β-glucan on yoghurt manufactured with Lactobacillus plantarum strains as adjunct culture. J. Food Agric. Environ. 11, 281–287. Kreisz, S., 2009. Malting. In: Esslinger, H.M. (Ed.), Handbook of Brewing: Processes, Technology, Markets. Wiley-VCH Verlag GmBH & Co., KGaA, Weinheim, pp. 147–164. Laitila, A., Sweins, H., Vilpola, A., Kotaviita, E., Olkku, J., Home, S., Haikara, A., 2006a. Lactobacillus and Pediococcus pentosaceus starter cultures as a tool for microflora management in malting and for enhancement of malt processability. J. Agric. Food Chem. 54, 3840–3851. Laitila, A., Wilhelmson, A., Kotaviita, E., Olkku, J., Home, S., Juvonen, R., 2006b. Yeast in an industrial malting ecosystem. J. Ind. Microbiol. Biotechnol. 33, 953–966. Linko, M., Haikara, A., Ritala, A., Penttila, O., 1998. Recent advances in the malting and brewing industry. J. Biotechnol. 65, 85–98. Lowe, D.P., Ulmer, H.M., Van Sinderen, D., Arendt, E.K., 2004. Application of biological acidification to improve the quality and processability of wort produced from 50 % raw barley. Inst. Guild Brew. 2, 133–140. Lowe, D.P., Arendt, E.K., Soriano, A.M., Ulmer, H.M., 2005. The influence of lactic acid bacteria on the quality of malt. J. Inst. Brew. 111, 42–50. Miller, G.L., Blum, R., Glennon, W.E., Burton, A.L., 1960. Measurement of carboxymethylcellulase activity. Anal. Biochem. 2, 127–132. Moraïs, S., Shterzer, N., Grinberg, I.R., Mathiesen, G., Eijsink, V.G.H., Axelsson, L., Lamed, R., Bayer, E.A., Mizrahi, I., 2013. Establishment of a simple Lactobacillus plantarum cell consortium for cellulase-xylanase synergistic interactions. Appl. Environ. Microbiol. http://dx.doi.org/10.1128/AEM.01211-13. Noots, I., Delcour, J.A., Michiels, C.W., 1999. From field barley to malt: detection and specification of microbial activity for quality aspects. Crit. Rev. Microbiol. 25, 121–153. Noots, I., Derycke, V., Michiels, C., Delcour, J.A., Delrue, R., Coppens, T., 2001. Improvement of malt modification by use of Rhizopus VII as starter culture. J. Agric. Food Chem. 49, 3718–3724. Okano, K., Zhang, Q., Yoshida, S., Tanaka, T., Ogino, C., Fukuda, H., Kondo, A., 2010. D-lactic acid production from cello-oligosaccharides and β-glucan using L-LDH gene-deficient and endoglucanase-secreting Lactobacillus plantarum. Appl. Environ. Microbiol. 85, 643–650. Rossi, F., Rudella, A., Marzotto, M., Dellaglio, F., 2001. Vector-free cloning of a bacterial endo-1,4-β-glucanase in Lactobacillus plantarum and its effect on the acidifying activity in silage: use of recombinant cellulolytic Lactobacillus plantarum as silage inoculant. Antonie Van Leeuwenhoek 80, 139–147. Ruijssenaars, H.J., Hartmans, S., 2001. Plate screening methods for the detection of polysaccharide-producing microorganisms. Appl. Microbiol. Biotechnol. 55, 143–149. Sohail, M., Naseeb, S., Sherwani, S.K., Sultana, S., Aftab, S., Shahzad, S., Ahmad, A., Khan, S.A., 2009. Distribution of hydrolytic enzymes among native fungi: Aspergillus the predominant genus of hydrolase producers. Pak. J. Bot. 41, 2567–2582. Strauss, M.L.A., Jolly, N.P., Lambrechts, M.G., Van Rensburg, P., 2001. Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts. J. Appl. Microbiol. 91, 182–190. Subash, C.B., Gopinath, P.A., Azariah, H., 2005. Extracellular enzymatic activity profiles in fungi isolated from oil-rich environments. Mycoscience 46, 119–126. Ullrich, S.E., 2011. Barley: Production, Improvement, and Uses. Wiley-Blackwell, USA.
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Short communication
Malting of barley with combinations of Lactobacillus plantarum, Aspergillus niger, Trichoderma reesei, Rhizopus oligosporus and Geotrichum candidum to enhance malt quality M. Hattingh a, A. Alexander b, I. Meijering b, C.A. van Reenen a, L.M.T. Dicks a,⁎ a b
Department of Microbiology, University of Stellenbosch, Stellenbosch 7600, South Africa Southern Associated Maltsters (Pty) LTD, P.O. Box 27, Caledon 7230, South Africa
a r t i c l e
i n f o
Article history: Received 14 August 2013 Received in revised form 10 December 2013 Accepted 20 December 2013 Available online 28 December 2013 Keywords: Malting Lactobacillus plantarum Aspergillus Trichoderma Rhizopus Geotrichum
a b s t r a c t Good quality malt is characterised by the presence of high levels of fermentable sugars, amino acids and vitamins. To reach the starch-rich endosperm of the kernel, β-glucan- and arabinoxylan-rich cell walls have to be degraded. β-Glucanase is synthesized in vast quantities by the aleurone layer and scutellum during germination. Secretion of hydrolytic enzymes is often stimulated by addition of the plant hormone gibberellic acid (GA3) during germination. We have shown an enhanced β-glucanase and α-amylase activity in malt when germinating barley was inoculated with a combination of Lactobacillus plantarum B.S1.6 and spores of Aspergillus niger MH1, Rhizopus oligosporus MH2 or Trichoderma reesei MH3, and L. plantarum B.S1.6 combined with cell-free culture supernatants from each of these fungi. Highest malt β-glucanase activity (414 Units/kg malt) was recorded with a combination of L. plantarum B.S1.6 and spores of A. niger MH1. Highest α-amylase activities were recorded with a combination of L. plantarum B.S1.6 and spores of R. oligosporus MH2 (373 Ceralpha Units/g malt). Highest FAN levels were recorded when L. plantarum was inoculated in combination with spores of either R. oligosporus MH2 or T. reesei MH3 (259 and 260 ppm, respectively). This is the first study showing that cell-free culture supernatants of Aspergillus, Rhizopus and Trichoderma have a stimulating effect on β-glucanase and α-amylase production during malting. A combination of L. plantarum B.S1.6, and spores of A. niger MH1 and R. oligosporus MH2 may be used as starter cultures to enhance malt quality. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Barley malt is the predominant raw material used in beer production (Haraldsson et al., 2004) and the quality thereof is characterised by the presence of high levels of fermentable sugars, amino acids and vitamins (Justé et al., 2011; Kreisz, 2009; Laitila et al., 2006a). During germination of barley, β-glucan is degraded by β-glucanase, synthesized in vast quantities by the aleurone layer and scutellum (Hrmova and Fincher, 2001). This leads to the production of (1,3;1,4)-β-D-triand tetrasaccharides, although oligosaccharides of up to 10 units may also form. Slow, or inadequately degraded β-glucan results in a high polymer content that leads to a hazy malt (Jin et al., 2004), high viscosity and poor lautering performance (Ullrich, 2011). To enhance the malting process, brewers often add gibberellic acid (GA3) to barley to break dormancy and reduce germination time (Kreisz, 2009; Noots et al., 1999). The addition of GA3, however, has to be carefully controlled. Excessive levels facilitate extensive rootlet formation and may lead to abnormally high sugar and soluble nitrogen levels, which in turn leads to abnormal colour development (Fox et al., ⁎ Corresponding author. Tel.: +27 21 808 5849. E-mail address: [email protected] (L.M.T. Dicks). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.12.017
2003). This led scientists searching for alternative methods to enhance germination (Goode and Arendt, 2006). Acidification of barley mash with Lactobacillus amylovorus improved fermentability and FAN content, and reduced β-glucan levels in wort (Lowe et al., 2004, 2005). This was attributed to the lowering of pH and the production of proteolytic and amylolytic enzymes. Laitila et al. (2006a) reported a reduction in wort viscosity and β-glucan content, and also an increase in xylanase and β-glucanase activities when mash was inoculated with Lactobacillus plantarum and Pediococcus pentosaceus. In other studies, where combinations of lactic acid bacteria and Geotrichum candidum were used, malt quality improved by restricting the growth of unwanted contaminants (Biovin and Malanda, 1997; Laitila et al., 2006b; Linko et al., 1998; Lowe et al., 2005). In one study a Rhizopus oligosporus starter culture enhanced malting (Noots et al., 2001). Apart from the possible production of proteolytic and amylolytic enzymes, lactic acid bacteria are known to produce antimicrobial peptides (bacteriocins) and antifungal compounds that may restrict the growth of bacteria and fungi. From these and similar studies (Foszcynska et al., 2004; Haikara et al., 1993; Noots et al., 1999), it is clear that the development of starter cultures containing lactic acid bacteria, separately or combined with fungi, may be an attractive alternative to chemical and enzymatic treatment of mash.
M. Hattingh et al. / International Journal of Food Microbiology 173 (2014) 36–40
The aim of this study was to determine whether strains of L. plantarum and spores of Aspergillus niger, R. oligosporus or Trichoderma reesei, originally isolated from barley, and G. candidum isolated from barley malt, could be used as starter cultures to enhance the malting process. To evaluate the performance of these strains during malting, the activity of α-amylase, β-glucanase, xylanase, cellulose and proteases, and the level of free amino nitrogen (FAN) content, was tested. 2. Material and methods 2.1. Microbial strains L. plantarum B.S1.6, A. niger MH1, T. reesei MH3 and R. oligosporus MH2 were originally isolated from barley. L. plantarum B.S1.6 was cultured in De Man, Rogosa and Sharpe (MRS) broth (Biolab, Biolab Diagnostics, Midrand, South Africa) at 30 °C, and strains MH1, MH3 and MH2 in Yeast Peptone Dextrose (YPD) medium (Biolab) at 26 °C. Geotrichum strains, isolated from barley malt, were identified as G. candidum based on sequencing of the D1/D2 region of the 26S rDNA (of rRNA gene), amplified with forward primer F63 (5′-GCATATCAAT AAGCGGAGGAAAAG-3′) and reverse primer LR3 (5′-GGTCCGTGTTTC AAGACGG-3′). The method of Garner et al. (2010) was used.
37
Actively growing cells of A. niger MH1, T. reesei MH3, R. oligosporus MH2 and the 12 strains of G. candidum were inoculated into Erlenmeyer flasks with 50 mL SC-medium, supplemented with either 1.0% (w/v) low viscosity CMC, 0.1% (w/v) β-glucan, 0.5% (w/v) beechwood xylan or 2.0% (w/v) starch. Incubation was on a rotary shaker (250 rpm) at 26 °C for 7 days. The cells were harvested (10,000 ×g, 15 min, 4 °C) and enzyme activities determined in the cfs. Cellulase and β-glucanase activities were determined by adding 20 μL 0.1% (w/v) CMC and 0.1% (w/v) β-glucan, each suspended in 0.05 M citrate buffer (pH 4.8), to 30 μL cfs in respective wells of a microtitre plate. The microtitre plate was incubated at 50 °C for 30 min and enzymatic reactions stopped by the addition of 100 μL DNS solution (1.0%, v/v, 3,5-dinitrosalicyclic acid; 20.0%, w/v, potassium sodium tartrate; 1.0%, w/v, NaOH; 0.2%, v/v, phenol and 0.05%, w/v, Na2SO3), followed by boiling for 15 min. The samples were allowed to cool to 25 °C and readings were taken at 540 nm (Bio Rad, Smartspec Plus). The same protocol was used to determine xylanase activity, except that 6 μL cfs was added to 54 μL of 1.0% (w/v) beechwood xylan, suspended in 0.05 M citrate buffer, pH 4.8 (Miller et al., 1960). The samples were incubated for 60 min and the reaction stopped by the addition of 90 μL DNS solution. Activity was expressed in nKats/mL. α-Amylase was determined using the Ceralpha method and activity expressed as CU/mL. The G. candidum strain with the highest overall enzyme activities was selected for further studies.
2.2. Enzyme activity assays 2.3. Preparation of starter cultures 2.2.1. Screening of cultures for the production of enzyme activity L. plantarum B.S1.6 was cultured in MRS broth at 30 °C for 8 h. Five microlitres of the culture was used to inoculate the following: MRS agar, supplemented with 1.4% (w/v) skimmed milk powder (Difco, Becton, Dickinson and Company, Sparks, Maryland, USA), 1.0% (w/v) carboxymethylcellulose (CMC) (Sigma-Aldrich), 0.1% (w/v) β-glucan (Difco), 0.5% (w/v) beechwood xylan (Carl Roth GmbH, Karlsruhe, Germany) or 2% (w/v) soluble starch (Merck, Darmstadt, Germany). After 48 h of incubation at 30 °C, substrate hydrolysis was detected as clear zones by positive colonies through flooding the plates with appropriate dyes. Plates containing CMC, β-glucan and xylan as substrates were stained with 0.1% (w/v) Congo red (Sigma-Aldrich) for 15 min. Excess dye was rinsed off with 1 M NaCl. Enzyme activity was enhanced by destaining plates with 1 M HCL, as described by Ruijssenaars and Hartmans (2001). Starch hydrolysis was observed by flooding the plates with iodine, while proteolytic zones were enhanced by adding 10% (v/v) tannic acid. Protease, cellulase, β-glucanase, xylanase and α-amylase activities of A. niger MH1, T. reesei MH3, R. oligosporus MH2 and the 12 Geotrichum strains were tested using the following protocol. Protease activity was determined on 1.4% (w/v) skimmed milk agar, cellulase activity on 0.67% (w/v) YNB agar (Sigma-Aldrich) supplemented with 1.0% (w/v) CMC, β-glucanase activity on YPD agar supplemented with 0.1% (w/v) β-glucan, as described by Strauss et al. (2001), xylanase activity on Potato Dextrose agar (PDA, Biolab) supplemented with 0.5% (w/v) beechwood xylan, and α-amylase on Nutrient agar (Biolab) and YNB agar (0.67%), each supplemented with 2.0% (w/v) soluble starch, as described by Buzzini and Martini (2002). The plates were incubated at 26 °C for 7 days and then tested for enzyme activity as described before. 2.2.2. Quantification of enzyme activities Enzyme reactions recorded with plate assays were quantified using spectrophotometric methods. Actively growing cells of L. plantarum B.S1.6 were inoculated into 50 mL MRS broth, supplemented with 2% (w/v) starch. After 40 h of incubation at 30 °C, the cells were harvested (10 000 ×g, 15 min, 4 °C) and 30 μL of each cell-free culture supernatant (cfs) added to wells in a 96-well microtitre plate. The plate was pre-incubated for 5 min at 50 °C. α-Amylase was determined using the Ceralpha method (Megazyme, International Ireland, Ltd, County Wicklow, Ireland) and activity expressed as Ceralpha units (CU) per mL.
L. plantarum B.S1.6 was cultured in MRS broth at 30 °C for 48 h, the cells harvested (10,000 ×g, 10 min, 10 °C) and resuspended in sterile distilled water to yield 8 × 106 cfu/20 mL. R. oligosporus MH2 and A. niger MH1 were streaked onto PDA and T. reesei MH3 onto Malt Extract Agar (MEA, Biolab), and incubated at 26 °C for 7 days. G. candidum strain 1173 was streaked onto YPD agar and incubated at 26 °C for 48 h. Spores of Rhizopus sp. MH2, T. reesei MH3, A. niger MH1 and G. candidum 1173 were washed from plates with 5 mL sterile physiological saline, counted using a haemocytometer (Paul Marienfeld GmbH & Co, Germany) and 8 × 108 spores suspended into 20 mL sterile distilled water. 2.4. Preparation of cell-free supernatant R. oligosporus MH2, T. reesei MH3 and A. niger MH1 were each cultured in 100 mL YNB, supplemented with 1.0% (w/v) low viscosity CMC, 0.1% β-glucan, 0.5% (w/v) beechwood xylan and 2.0% (w/v) starch, respectively. All cultures were incubated aerobically, on a rotary shaker (250 rpm) at 26 °C for 7 days. Cell-free culture supernatants were collected separately by filtration through sterile 0.22 μm nitrocellulose membranes. Cellulase, β-glucanase and xylanase activities were determined using the DNS-assay and α-amylase activity using the Ceralpha method, as described elsewhere. Twenty microlitres of the cfss of A. niger MH1, T. reesei MH3 and Rhizopus sp. MH2 was used to inoculate the barley. 2.5. Malting Eight micro-malting trials were conducted in separate 5 L plastic containers (strains MH1 + B.S1.6, MH2 + B.S1.6, MH3 + B.S1.6, 1173 + B.S1.6, MH1 cfs + B.S1.6, MH2 cfs + B.S1.6, MH3 cfs + B.S1.6 and the control, not inoculated with cells or cfs). Barley (500 g, Erica cultivar) was added to a 5 L plastic container with holes (1 mm in diameter) drilled into the bottom of the container. The container was placed inside an empty 5 L container linked to an air pump. Sterile distilled water (800 mL) was added to the barley in the inner container. The barley kernels were steeped for 43 h (9 h wet stand, followed by 14 h air rest, 14 h wet stand and 6 h air rest), germinated for 84 h and then kilned in five stages (the length of each stage: 3, 5, 12, 4 and 2 h). At the start of germination, the barley kernels were inoculated
38
M. Hattingh et al. / International Journal of Food Microbiology 173 (2014) 36–40
Table 1 Hydrolysis of casein, cellulose, β-glucan, xylan and starch (enzyme activities are shown in parenthesis). Strain
Hydrolysis of:
L. plantarum B.S1.6 A. niger MH2 T. reesei MH3 Rhizopus oligosporus MH2 G. candidum 1173 a b
Casein
Cellulosea
β-Glucana
Xylana
Starchb
− − − − +
− (4.7 ± 0.4) (17.6 ± 2.7) (0.8 ± 0.1) (0.5 ± 0.3)
− (7.4 ± 0.7) (10.9 ± 0.6) (0.6 ± 0.1) (0.4 ± 0.1)
− (217.6 ± 12.7) (53.9 ± 4.3) (0.6 ± 0.2) (0.9 ± 0.2)
+ (33.7 ± 5.2) (171.0 ± 8.11) −
Enzyme activity in nkats/mL. Amylase activity in CU/mL.
with 20 mL L. plantarum B.S1.6 (8 × 106 cfu) and 20 mL (8 × 108 spores) of either A. niger MH1, T. reesei MH3, R. oligosporus MH2 or G. candidum 1173. This corresponded to 1.6 × 104 cfu L. plantarum B.S1.6 and 1.6 × 106 spores per gramme barley. After germination, samples were collected and viable cell numbers of L. plantarum B.S1.6 determined by plating onto MRS agar. Cell numbers of A. niger MH1, T. reesei MH3 and R. oligosporus MH2 were determined by plating onto MEA, and cell numbers of G. candidum 1173 by plating onto YPD. All plates were incubated for 48 h at 26 °C. In another experiment, 20 mL cfs of either strain MH1, MH3 or MH2 were added to 20 mL L. plantarum B.S1.6. The control received 20 mL sterile distilled water. Germination was for 84 h, followed by kilning for 26 h. 2.6. Malt analyses Kilned malt was analysed according to methods used by the European Brewing Convention (Bamforth and Barclay, 1993). In brief, this entailed grinding of 50 g kilned malt in a disc mill (Bühler-Miag 4000, Bühler Ltd., Milan, Italy) with pore sizes of 0.2 mm. Distilled water (400 mL) was added to the grinded malt and the suspension placed in a mash bath (Industrial Equipment Corporation). The mash was filtered and the β-glucan and free-amino nitrogen (FAN) content in the wort determined according to methods used by the American Society of Brewing Chemists (2009). Beta-glucanase activity in the malt was determined using the Azo-barley glucan method (K-MBGL 03/11, Megazyme). Reactions were conducted at 30 °C and 60 °C to differentiate between malt β-glucanase and microbial β-glucanase activity, respectively (Home et al., 1993; Laitila et al., 2006a). Xylanase activity was determined using the Xylazyme AX method (T-XAX200 10/2008, Megazyme). Xylanase enzyme extraction was performed for 15 min at room temperature in sodium acetate buffer (25 mM, pH 4.7), with continuous stirring. α-Amylase activity was quantified with the Ceralpha kit (K-CERA 08/05, Megazyme), as described before. 2.7. Statistical analyses Statistical analyses were done using Statistica v. 10 (StatSoft, Inc.). The students' T-test was performed at 95% confidence levels. Significant differences (p = 0.05) were indicated by an asterisk (*).
3. Results and discussion 3.1. Enzyme production and activities Hydrolysis of casein, cellulose, β-glucan, xylan and starch, and activity of α-amylase is listed in Table 1. The hydrolysis of starch and α-amylase by L. plantarum is characteristic of the species (Hammes and Hertel, 2009). Most lactic acid bacteria, including L. plantarum, do not have proteolytic, cellulase, β-glucanase or xylanase activities (Hammes and Hertel, 2009; Okano et al., 2010). However, a few exceptions have been reported. Khalid and Marth (1990) described the strains of L. plantarum with the ability to hydrolyse casein, and Kilic and Akpinar (2013) have shown that low concentrations of β-glucan (0.5% and 1.0%, w/v) enhanced the growth of probiotic strains of L. plantarum. Attempts to genetically engineer strains of L. plantarum to degrade xylan, cellulose and β-glucan have been successful (Moraïs et al., 2013; Rossi et al., 2001). A. niger MH1, T. reesei MH3 and R. oligosporus MH2 did not hydrolyse casein on skimmed milk plates and no proteolytic activity was detected in the cfs (Table 1). Not all strains of Aspergillus, Trichoderma and Rhizopus hydrolyse casein, as shown by Sohail et al. (2009). Of the nine proteolytic G. candidum isolates, strain 1173 formed the largest zone of casein hydrolysis. Casein hydrolysis is a well-documented characteristic of G. candidum (Ahearn et al., 1968). A. niger MH1, T. reesei MH3, R. oligosporus MH2 and all 12 strains of G. candidum hydrolysed cellulose and β-glucan on plates and would thus be able to degrade the cell walls protecting the starch-rich grains in the endosperm. A. niger MH1, T. reesei MH3, R. oligosporus MH2, and three of the 12 G. candidum isolates, including strain 1173, hydrolysed xylan (Table 1). Degradation of xylan is common amongst fungi (Sohail et al., 2009). A. niger MH1 and R. oligosporus MH2 hydrolysed starch on plates and produced α-amylase at 33.66 ± 5.23 and 171 ± 8.11 CU/mL, respectively, in cfs (Table 1). Amylase production by Aspergillus and Rhizopus spp. is well reported (Sohail et al., 2009). Starch hydrolysis by Geotrichum spp., on the other hand, is less common and only a few strains with amylase activity have been reported. Arotupin (2007) isolated an amylase-positive strain of G. candidum from Cassava waste water and Falih (1998) described two amylase-producing G. candidum strains isolated from soil. Of the 10 Geotrichum klebahnii strains described by Buzzini and Martini (2002), only one produced an amylase.
Table 2 Enzyme activities in malt produced with L. plantarum B.S1.6, in combination with viable cells and cell-free supernatant (cfs) of A. niger MH1, R. oligosporus MH2, T. reesei MH3 and G. candidum. Significantly increased enzyme activities are indicated by*. Malting trial
α-Amylase (CU/g malt)
β-Glucanase (U/kg malt at 30 °C)
β-Glucanase (U/kg malt at 60 °C)
Total β-glucanase (U/kg at 30 °C + 60 °C)
Xylanase (U/g malt)
A. niger MH1 R. oligosporus MH2 T. reesei MH3 G. candidum 1173 niger MH1 cfs R. oligosporus MH2 cfs T. reesei MH3 cfs Control
355* 373* 338* 166 314* 365* 346* 151
414* 282* 347* 207 324* 343* 293 155
274* 203* 228* 131 201* 243* 189* 78
688* 485* 575* 338 525* 586* 482* 232
0.29 0.33 0.30 0.25 0.39* 0.46* 0.37* 0.22
M. Hattingh et al. / International Journal of Food Microbiology 173 (2014) 36–40
L. plantarum B.S1.6 and spores of T. reesei MH3 decreased the β-glucan in malt from 60 ppm to 54 and 49 ppm, respectively (Fig. 1). The greater decrease in β-glucan content in the presence of L. plantarum B.S1.6 and T. reesei MH3 co-insides with the high β-glucanase activity recorded in the cfs of strain MH3 (10.85 ± 0.56). Less β-glucan was degraded when barley was inoculated with a combination of L. plantarum B.S1.6 and spores of R. oligosporus MH2, as indicated by a decrease from 60 ppm to 57 ppm (Fig. 1). A similar poor degradation of β-glucan (from 60 ppm to 59 ppm) was recorded when barley was inoculated with a combination of L. plantarum B.S1.6 and G. candidum 1173 (Fig. 1). This corresponds with the weak β-glucanase activities recorded in the cfs of these strains. Barley inoculated with L. plantarum B.S1.6 and spores of A. niger MH1, or L. plantarum B.S1.6 and spores of T. reesei MH3 had the highest total β-glucanase activities when measured at 30 °C and 60 °C (688 and 575 U/kg malt, respectively; Table 2). The overall β-glucanase activity recorded when barley was inoculated with L. plantarum B.S1.6 and spores of Rhizopus sp. MH2 was much lower (485 U/kg malt, Table 2). Aspergillus spp. and Rhizopus spp. are used as starter cultures in Asian fermented cereals. Rhizopus spp. has been used to enhance malt modification (Biovin and Malanda, 1997). L. plantarum B.S1.6 was selected based on its ability to produce cell-bound and extracellular α-amylase (Hattingh, 2013). The total β-glucanase activities (malt and microbial enzyme activities) produced during malting by starter cultures containing spores of A. niger MH1 or T. reesei MH3 were higher than β-glucanase activities produced in the presence of cfss of Aspergillus sp. MH1 and T. reesei MH3 (Table 2). However, much higher β-glucanase activities were recorded in malt inoculated with cfs of R. oligosporus MH2 (586 U/kg malt) compared to spores (485 U/kg malt, Table 2). The FAN content reflects the extent at which proteolysis takes place during malting and mashing. A high FAN content is essential to ensure active fermentation by the brewers' yeast (Lowe et al., 2004). The FAN content of kilned malt prepared without starter cultures or cfs was 205 ppm and much lower compared to the FAN content recorded when barley was inoculated with starter cultures or cfss (Fig. 2). Highest FAN levels were recorded when L. plantarum was inoculated in combination with spores of either Rhizopus sp. MH2 or T. reesei MH3 (259 and 260 ppm, respectively; Fig. 2). Slightly higher FAN levels were recorded when cfs of A. niger MH1 or R. oligosporus MH2 (267 ppm; Fig. 2) were used. The latter combination is thus preferred. Highest increase in α-amylase activity (373 CU/g malt) was recorded when malting was performed in the presence of L. plantarum B.S1.6 and
70 60 50 *
* *
40 30 20 10 0 Aspergillus Rhizopus sp. sp. MH1 MH2
T. reesei MH3
G. candidum Aspergillus Rhizopus sp. 1173 sp. MH1 cfs MH2 cfs
T. reesei MH3 cfs
39
Control
Malting trial Fig. 1. Beta-glucan content of kilned malt from seven malting trials and the control. Lactobacillus plantarum B.S1.6 was combined with the respective starter cultures. Significant changes in β-glucan content are indicated by *.
None of the Geotrichum isolates studied by Laitila et al. (2006b), Noots et al. (2001) and Subash et al. (2005) degraded starch. Of the 12 G. candidum isolates tested, strain 1173 produced the highest level of cellulose, β-glucanase and xylanase activities and was selected as starter culture. 3.2. Malting The cell numbers of L. plantarum B.S1.6 decreased from 1.6 × 104 cfu to 1 × 104 cfu per gramme malt and the spore count of A. niger MH1, T. reesei MH3 and R. oligosporus MH2 from 1.6 × 106 cfu to 1.0 × 104 cfu per gramme malt after 84 h of germination. The cell numbers of G. candidum 1173 decreased from 1.6 × 106 cfu to 1.0 × 104 cfu during the same period. The total β-glucanase activity in the control malt, i.e. β-glucanase recorded for malt (readings taken at 30 °C) plus β-glucanase related to microbial activity (readings taken at 60 °C), was 233 U/kg malt (Table 2). In all experiments with control malt the β-glucanase activity pertaining to microbial activity was always lower, suggesting that microorganisms naturally present in barley contribute very little to the production of β-glucanase. Inoculation of barley with a combination of L. plantarum B.S1.6 and spores of A. niger MH1, or
* 280 260
*
*
* *
240
FAN (ppm)
*
*
220 200 180 160 140 120 100 Aspergillus Rhizopus sp. MH1 sp. MH2
T. reesei MH3
G. Aspergillus Rhizopus T. reesei candidum sp. MH1 cfs sp. MH2 cfs MH3 cfs 1173
Control
Malting trial Fig. 2. Free amino nitrogen (FAN) content of kilned malt recorded for seven malting trials and the control. Lactobacillus plantarum B.S1.6 was combined with the respective starter cultures. Significant changes in FAN content are indicated by *.
40
M. Hattingh et al. / International Journal of Food Microbiology 173 (2014) 36–40
spores of R. oligosporus MH2 (Table 2). Xylanase activities were very low and compared to that recorded in the control malting trial (Table 2). 4. Conclusion Concluded from the seven malting trials, β-glucan was most actively degraded in the presence of L. plantarum B.S1.6 and cfs of A. niger MH1, Rhizopus sp. MH2 and T. reesei MH3. Highest FAN levels were recorded when L. plantarum was inoculated in combination with spores of either R. oligosporus MH2 or T. reesei MH3. Malting could be enhanced by inoculating barley with a combination of L. plantarum B.S1.6, A. niger MH1, R. oligosporus MH2 and T. reesei MH3. Large scale brewing experiments will have to be conducted to evaluate the starter cultures. Acknowledgements The National Research Foundation (NRF) of South Africa for a bursary to M. Hattingh is acknowledged. References Ahearn, D.G., Meyers, S.P., Nichols, R.A., 1968. Extracellular proteinases of yeasts and yeastlike fungi. Appl. Microbiol. 16, 1370–1374. American Society of Brewing Chemists, 2009. Methods of analysis, Statistical Analysis-2 Limits of Detection and Determination2009 edition. The Society, St. Paul, MN. Arotupin, D.J., 2007. Evaluation of microorganisms from cassava waste water for production of amylase and cellulase. Res. J. Microbiol. 2, 475–480. Bamforth, C.W., Barclay, A.H.P., 1993. Malting technology and the use of malt. In: MacGregor, A.W., Bhatty, R.S. (Eds.), Barley: Chemistry and Technology. American Association of Cereal Chemists, St. Paul, Minnesota. Biovin, P., Malanda, M., 1997. Improvement of malt quality and safety by adding starter culture during the malting process. Master Brew. Assoc. Am. 34, 96–101. Buzzini, P., Martini, A., 2002. Extracellular enzyme activity profiles in yeast and yeast-like strains isolated from tropical environments. J. Appl. Microbiol. 93, 1020–1025. Falih, A.M., 1998. Effect of heavy-metals on amylolytic activity of the soil yeasts Geotrichum captatum and Geotrichum candidum. Bioresour. Technol. 66, 213–217. Foszcynska, B., Dzubia, E., Stempniewicz, R., 2004. The use of Geotrichum candidum starter culture for protection of barley and its influence on biotechnological qualities of malts. Electron. J. Pol. Agric. Univ. 7. Fox, G.P., Panozzo, J.F., Li, C.D., Lance, R.C.M., Inkerman, P.A., Henry, R.J., 2003. Molecular basis of barley qualities. Aust. J. Agric. Res. 54, 1081–1101. Garner, C.D., Starr, J.K., McDough, P.L., Altier, C., 2010. Molecular identification of veterinary yeast isolates by use of sequence-based analysis of the D1/D2 region of the large ribosomal subunit. J. Clin. Microbiol. 48, 2140–2146. Goode, D.L., Arendt, E.K., 2006. Development in the supply of adjunct materials for brewing. In: Bamforth, C.W. (Ed.), Brewing, New Technologies. Woodhead Publishing Ltd, Cambridge, England, pp. 30–58. Haikara, A., Uljas, H., Suurnaki, A., 1993. Lactic acid starter cultures in malting — a novel solution to gushing problems. Proceedings of the European Brewery Convention Congress, Lisbon, 24. IRL Press, Oxford, pp. 163–172. Hammes, W.P., Hertel, C., 2009. Genus I. Lactobacillus Beijerinck 1901, 212AL, In: De Vos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A., Schleifer, K.-H., Whitman, W.B. (Eds.), Bergey's manual of systematic bacteriology, 2nd edition. The Firmicutes, vol. 3. Springer, New York, pp. 465–511. Haraldsson, A., Rimsten, L., Alminger, M.L., Andersson, R., Andid, T., Aman, P., Sandberg, A., 2004. Phytate content is reduced and β-glucanase activity suppressed in malted barley steeped with lactic acid at high temperature. J. Sci. Food Agric. 84, 653–662.
Hattingh, M., 2013. The Effect of Lactic Acid Bacteria and Fungi on the Malting of Barley. (MSc thesis) University of Stellenbosch, Stellenbosch, South Africa. Home, S., Pietila, K., Sjöholm, K., 1993. Control of glucanolysis on mashing. J. Am. Soc. Brew. Chem. 51, 108–113. Hrmova, M., Fincher, G.B., 2001. Structure-function relationships of β-D-glucan endo- and exohydrolyses from higher plants. Plant Mol. Biol. 47, 73–91. Jin, Y.L., Speers, R.A., Paulson, A.T., Stewart, R.J., 2004. Barley β-glucans and their degredation during malting and brewing. Master Brew. Assoc. Am. Tech. Q. Pap. 41, 231–240. Justé, A., Malfliet, S., Lenaerts, M., de Cooman, L., Aerts, G., Willems, K.A., Lievens, B., 2011. Microflora during malting of barley: overview and impact on malt quality. Brew. Sci. 64, 22. Khalid, N., Marth, E.H., 1990. Proteolytic activity by strains of Lactobacillus plantarum and Lactobacillus casei. J. Dairy Sci. 73, 3068–3076. Kilic, G.B., Akpinar, D., 2013. The effects of different levels of β-glucan on yoghurt manufactured with Lactobacillus plantarum strains as adjunct culture. J. Food Agric. Environ. 11, 281–287. Kreisz, S., 2009. Malting. In: Esslinger, H.M. (Ed.), Handbook of Brewing: Processes, Technology, Markets. Wiley-VCH Verlag GmBH & Co., KGaA, Weinheim, pp. 147–164. Laitila, A., Sweins, H., Vilpola, A., Kotaviita, E., Olkku, J., Home, S., Haikara, A., 2006a. Lactobacillus and Pediococcus pentosaceus starter cultures as a tool for microflora management in malting and for enhancement of malt processability. J. Agric. Food Chem. 54, 3840–3851. Laitila, A., Wilhelmson, A., Kotaviita, E., Olkku, J., Home, S., Juvonen, R., 2006b. Yeast in an industrial malting ecosystem. J. Ind. Microbiol. Biotechnol. 33, 953–966. Linko, M., Haikara, A., Ritala, A., Penttila, O., 1998. Recent advances in the malting and brewing industry. J. Biotechnol. 65, 85–98. Lowe, D.P., Ulmer, H.M., Van Sinderen, D., Arendt, E.K., 2004. Application of biological acidification to improve the quality and processability of wort produced from 50 % raw barley. Inst. Guild Brew. 2, 133–140. Lowe, D.P., Arendt, E.K., Soriano, A.M., Ulmer, H.M., 2005. The influence of lactic acid bacteria on the quality of malt. J. Inst. Brew. 111, 42–50. Miller, G.L., Blum, R., Glennon, W.E., Burton, A.L., 1960. Measurement of carboxymethylcellulase activity. Anal. Biochem. 2, 127–132. Moraïs, S., Shterzer, N., Grinberg, I.R., Mathiesen, G., Eijsink, V.G.H., Axelsson, L., Lamed, R., Bayer, E.A., Mizrahi, I., 2013. Establishment of a simple Lactobacillus plantarum cell consortium for cellulase-xylanase synergistic interactions. Appl. Environ. Microbiol. http://dx.doi.org/10.1128/AEM.01211-13. Noots, I., Delcour, J.A., Michiels, C.W., 1999. From field barley to malt: detection and specification of microbial activity for quality aspects. Crit. Rev. Microbiol. 25, 121–153. Noots, I., Derycke, V., Michiels, C., Delcour, J.A., Delrue, R., Coppens, T., 2001. Improvement of malt modification by use of Rhizopus VII as starter culture. J. Agric. Food Chem. 49, 3718–3724. Okano, K., Zhang, Q., Yoshida, S., Tanaka, T., Ogino, C., Fukuda, H., Kondo, A., 2010. D-lactic acid production from cello-oligosaccharides and β-glucan using L-LDH gene-deficient and endoglucanase-secreting Lactobacillus plantarum. Appl. Environ. Microbiol. 85, 643–650. Rossi, F., Rudella, A., Marzotto, M., Dellaglio, F., 2001. Vector-free cloning of a bacterial endo-1,4-β-glucanase in Lactobacillus plantarum and its effect on the acidifying activity in silage: use of recombinant cellulolytic Lactobacillus plantarum as silage inoculant. Antonie Van Leeuwenhoek 80, 139–147. Ruijssenaars, H.J., Hartmans, S., 2001. Plate screening methods for the detection of polysaccharide-producing microorganisms. Appl. Microbiol. Biotechnol. 55, 143–149. Sohail, M., Naseeb, S., Sherwani, S.K., Sultana, S., Aftab, S., Shahzad, S., Ahmad, A., Khan, S.A., 2009. Distribution of hydrolytic enzymes among native fungi: Aspergillus the predominant genus of hydrolase producers. Pak. J. Bot. 41, 2567–2582. Strauss, M.L.A., Jolly, N.P., Lambrechts, M.G., Van Rensburg, P., 2001. Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts. J. Appl. Microbiol. 91, 182–190. Subash, C.B., Gopinath, P.A., Azariah, H., 2005. Extracellular enzymatic activity profiles in fungi isolated from oil-rich environments. Mycoscience 46, 119–126. Ullrich, S.E., 2011. Barley: Production, Improvement, and Uses. Wiley-Blackwell, USA.
