1605 Journal of Food Protection, Vol. 77, No. 9, 2014, Pages 1605-1611 doi: 10.4315/0362-028X. JFP-14-145 Copyright © , International Association for Food Protection
Research Note
Metabolic Characterization of Bacillus subtilis and Bacillus amyloliquefaciens Strains Isolated from Traditional Dry-Cured Sausages AIDA CACHALDORA, SONIA FONSECA, MARIA GOM EZ, INMACULADA FRANCO, AND JAVIER CARBALLO* Area de Tecnologia de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain MS 14-145: Received 27 March 2014/Accepted 29 April 2014
ABSTRACT The aim of this study was to investigate the effect of pH, temperature, and NaCl on growth, proteolytic and lipolytic activities, and the ability to produce biogenic amines of 19 strains of Bacillus isolated from Androlla and Botillo (two Spanish traditional sausages) to elucidate the role of these bacteria in sausage manufacture. All strains grew in the presence of 10% salt and at pH values of 5.0 and 5.5, whereas only 9 strains grew at 10°C. Proteolytic activity was assessed by the agar plate method, which revealed that 100 and 94.7% of the strains were able to hydrolyze sarcoplasmic and myofibrillar proteins, respectively. These results were confirmed by electrophoretic assays. The titration method revealed that only two strains hydrolyzed pork fat to any extent, and the profiles of the fatty acids freed were different. Most strains produced biogenic amines, but the quantities were generally low.
Raw-fermented sausages are widely consumed meat products in which the microbial activity plays an important role during the manufacturing process. Microorganisms are responsible for the fermentation process and mainly responsible for the proteolytic and lipolytic changes that occur during the ripening of these products, which determines their sensorial characteristics (texture, aroma, and taste). The microbiota of traditional dry-cured sausages is composed of a wide variety of microorganisms from the raw materials and the environment (13, 16-18, 40). Two major groups of bacteria dominate most of the ripening time and play well-known roles: lactic acid bacteria and gram positive coagulase-negative cocci, mainly represented by Staphylococcus and Kocuria (8). Lactic acid bacteria guarantee the safety of the products through the production of antimicrobial compounds such as lactic acid and bacteriocins, and coagulase-negative cocci contribute to color stability and enhance sensory properties due to liberation and degradation of amino acids and fatty acids (41). However, other microbial groups such as enterobac teria, Bacillus, and molds and yeasts may also proliferate, but their specific roles are not well understood (20). The genus Bacillus consists of a diverse array of gram positive aerobic and facultative anaerobic spore-forming rods that can be isolated from a wide variety of sources. Many species are important as food-spoilage organisms and can contaminate fruits, vegetable products, milk and other * Author for correspondence. Tel: + 34-988-387052; Fax: + 34-988387001; E-mail: [email protected].
dairy products, spices, and meat and meat products; however, these bacteria also play an important role in production of fermented products because of their proteo lytic activity (24, 30, 42). Several Bacillus species (B. subtilis, B. amyloliquefa ciens, B. pumilus, B. circulans, and B. megaterium) have been isolated from traditional dry-cured sausages (3, 9, 30). The presence of Bacillus in fermented sausages seems to be important. In a recent study (14), Bacillus strains were the only microorganisms isolated on manitol salt agar from a fermented sausage (Galician chorizo) meat mix before stuffing, with counts of 4.99 log CFU/g. Bacillus in fermented sausages have proteolytic and lipolytic activities (3), which could complement the effects of autochthonous enzymes from meat, lactic acid bacteria enzymes, and coagulase-negative cocci enzymes during sausage ripening. Therefore, the presence of Bacillus should have an effect (positive or negative) on the organoleptic characteristics of the final products. Androlla and Botillo are two traditional sausages widely made and consumed in northwestern Spain (29). Bacillus microorganisms have not been counted directly in Androlla or Botillo sausages. However, mean counts on standard plate count agar plus 7.5% NaCl were 6.87 log CFU/g for Androlla sausages (17) and 6.56 log CFU/g for Botillo sausages (76); approximately 10% of the bacterial strains from these sausages isolated on the standard plate count agar plus 7.5% NaCl culture medium were later identified as Bacillus species. The aim of this study was to determine the growth and metabolic properties of 19 strains of Bacillus isolated from
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TABLE 1. Bacillus strain identity, source sausage, lipolytic activity assessed by titration method, and biogenic amine productiona Biogenic amines (ppm) Strain no. SA06 SA26 SA28 SA35 SA37 SA39 SA43 SA50 SB01 SB05 SB07 SB09 SB13 SB14 SB15 SB16 SB17 SB 18 SB26
Species
B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B.
subtilis amyloliquefaciens subtilis amyloliquefaciens amyloliquefaciens amyloliquefaciens amyloliquefaciens subtilis subtilis subtilis subtilis amyloliquefaciens subtilis subtilis subtilis subtilis subtilis subtilis subtilis
Source Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo
Lipolytic activity (% oleic acid) 0.22 0.94 1.26 1.17 0.76 1.08 1.34 0.15 0.04 0.45 13.86 0.15 21.19 0.26 1.64 1.03 0.90 0.47 0.31
(0.20-0.24) (0.92-0.96) (1.23-1.29) (1.14-1.20) (0.74-0.78) (1.06-1.10) (1.30-1.38) (0.12-0.18) (0.02-0.06) (0.10-0.80) (13.58-14.14) (0.12-0.18) (21.03-21.35) (0.04-0.48) (1.61-1.69) (0.84-1.22) (0.85-0.95) (0.47-0.47) (0.29-0.33)
Putrescine 18.43 1.22 0.70 0.76 2.49 2.27 3.27 1.19 1.27 0.57 0.39 1.33 0.86
(16.80-20.03) (1.22-1.22) (0.60-0.80) (0.68-0.84) (2.46-2.52) (2.25-2.29) (3.15-3.39) (1.19-1.19) (1.27-1.27) (0.56-0.58) (0.38-0.40) (1.16-1.50) (0.61-1.11) ND ND ND ND ND 1.75 (1.54-1.96)
Cadaverine 0.43 3.07 0.44 1.08 0.53 0.56 2.52 4.29 3.08 0.58 0.59 0.62 0.52
(0.41-0.45) (3.07-3.07) (0.31-0.57) (0.76-1.40) (0.34-0.72) (0.29-0.83) (2.51-2.53) (4.29-4.29) (3.08-3.08) (0.51-0.65) (0.32-0.86) (0.54-0.70) (0.39-0.65) ND ND ND ND ND 0.50 (0.44-0.56)
" Values are means (ranges) of two replicates. ND, not detected.
Androlla and Botillo sausages. This is the first step in selecting appropriate strains for further studies (inoculation of experimental sausages and analysis of their physico chemical, microbiological, and organoleptic characteristics), determining the role of Bacillus spp. in the ripening of the fermented sausages, and understanding the effect of this group of microorganisms on the quality of the final products.
Effect of pH, temperature, and NaCl on microbial growth. Each strain was tested for its ability to grow at 10°C in BHI broth at pH 7.0, in BHI broth adjusted to pH 5 and 5.5 with lactic acid, and in BHI broth supplemented with 10 and 15% NaCl. Ten microliters of an overnight culture of each strain was inoculated into 5 ml of these test media, and the growth was scored as positive or negative after incubation at 37°C for 72 h. Growth was scored as positive when tubes were more turbid than the noninoculated and incubated tubes used as control.
MATERIALS AND METHODS
Proteolytic activity: qualitative assessment by the agar plate method. Sarcoplasmic and myofibrillar proteins were extracted according to the method of Fadda et al. (10). Sarcoplasmic proteins were sterilized by filtration through a polyvinylidene fluoride filter (0.22-pm pore size; Millipore, Billerica, MA). Myofibrillar proteins were extracted under sterile conditions. The sarcoplasmic and myofibrillar proteins were added at concentrations of 0.5 and 0.2 mg/ml, respectively, to sterile medium containing 0.25% yeast extract, 0.1% glucose, and 1.5% agar. The medium was poured into petri dishes, and after solidification, three wells were bored in the agar in each plate, and 40 pi of cell suspension was pipetted into each well. After incubation at 37°C for 48 h, the agar disc was removed from each dish and stained for 30 min in 0.05% (wt/vol) Coomassie blue R-250 (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in methanol-acetic acid-water (50:10:40) and destained in methanolethanol-acetic acid-water (20:10:5:65). The diameter of the clear zone surrounding each inoculated well was recorded as an indication of proteolytic activity.
Bacillus strains and preparation of cell suspensions. The B. subtilis and B. amyloliquefaciens strains used in this study (Table 1) were isolated on standard plate count agar (Oxoid, Basingstoke, UK) plus 7.5% NaCl (16, 17). For the Androlla and Botillo sausages, 13 of the 200 isolates and 17 of the 150 isolates, respectively, found on standard plate count agar plus 7.5% NaCl were later determined to be Bacillus spp. Strains were identified by sequencing the 16S rRNA gene and comparing the obtained sequences with those available in the GenBank database (National Center for Biotechnology Information, Bethesda, MD). The genomic DNA extracted from the Bacillus isolates was then subjected to repetitive sequence-based PCR analysis using the single oligonucleotide primer (GTG)5 as described by Fonseca et al. (14). The 19 strains selected for this study had different (GTG)5-PCR fingerprinting profiles, indicating differences among strains of the same species. Strains were stored at —80°C in brain heart infusion (BHI) broth (Oxoid) with 20% (vol/vol) glycerol as a cryoprotective agent. To prepare the cell suspensions, a correlation between the log CFU per milliliter and the absorbance at 650 nm was established for each strain. Samples of BHI broth cultures were collected after 24 h of incubation, and the absorbance at 650 nm was measured. The cultures were then centrifuged at 12,000 x g for 10 min, and the resulting pellets were washed twice with 20 mM phosphate buffer pH 7.0 and then resuspended in the same buffer to obtain inocula containing approximately 109 CFU/ml.
Proteolytic activity: quantitative assessment by electro phoretic methods. Strains that exhibited proteolytic activity by the agar plate method were then tested by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Actin and myosin were extracted and quantified using the methods described by Perez-Juan et al. (34) and Abdel-Mohsen et al. (1), respectively, with modifications in the centrifugation times and composition of
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TABLE 2. Bacillus strain growth and metabolic characteristics No. of positive isolates Characteristic
B. subtilis
B. amyloliquefaciens
13 13 9
6 6 1
13 10
6 3
13 (11, halo > 25 mm; 2, halo < 25 mm) 13 (11, halo > 25 mm; 2, halo < 25 mm)
6 (halo > 25 mm) 5 (4, halo < 25 mm; 1, halo < 25 mm)
10 12 8 8
4 6 6 6
13
6
Growth at: pH 5.0 pH 5.5 10°C Growth in NaCl 10% 15% Proteolytic activity0 Sarcoplasmic proteins Myofibrillar proteins Decarboxylase activity0 Histidine Tyrosine Ornithine Lysine Total no. of isolates tested ° Determined with the agar plate method. the buffers for resuspension (12). A 0.2-ml volume of cell suspension was inoculated with 1 ml of sarcoplasmic protein, actin, and myosin extract (0.15 mg/ml) supplemented with 1% glucose and incubated at 37°C for 72 h in a shaking incubator. Uninoculated protein extracts were used as controls and incubated under the same conditions. After incubation, samples were examined to determine proteolytic activity. Protein degradation was assayed by SDS-PAGE as described by Laemmli (27) on 12% polyacrylamide gels. Ten microliters of each protein preparation was mixed with 19 ml of Laemmli buffer (Bio-Rad, Hercules, CA) and 1 ml of [1-mcrcaptoethanol, and 30 pi of each preparation and 10 pi of SDS-PAGE molecular weight standard low range (BioRad) were placed in different wells in the gel. Electrophoresis was carried out at 220 V for about 45 min. Gels were soaked in a fixing solution of methanol-water-acetic acid (50:43:7) for 15 min, stained in 0.05% (wt/vol) Coomassie blue R-250 in methanolacetic acid-water (45:10:45) for 2 h, and destained until the background was clear. The molecular weights of the products of proteolysis were estimated by reference to the relative mobilities of standard proteins. The results corresponding to each band were expressed as a percentage of total absorbance at 550 nm calculated with Quantity One Software (Bio-Rad).
Lipolytic activity. Lipolytic activity was assessed using the method described by Vignolo et al. (43) with some modifications. One milliliter of a cell suspension of each strain was inoculated into 30 ml of a sterile broth containing 1% (wt/vol) peptone, 0.5% (wt/vol) yeast extract, 0.5% (wt/vol) meat extract, and 3% (wt/vol) NaCl, pH 7.0, supplemented with 15 g of pork fat. After incubation at 37°C for 72 h with shaking, the lipolytic activity was measured by titration. The fat was extracted according to the method of Folch et al. (11), and the free fatty acids were titrated following the procedure of Vignolo et al. (43). The free fatty acid concentration was expressed as percentage of oleic acid (31). The same medium was also used to determine the ability of the strains to release free fatty acids from triglycerides. After 72 h of incubation, the fat in each culture was extracted as described, and the free fatty acids were separated from the triglycerides in
columns of NH2-aminopropyl following the procedure described by Kaluzny et al. (26). Methyl esters of free fatty acids were prepared using the method of Shehata et al. (38) and were quantified by gas chromatography following the procedure described by Franco et al. (15) using an internal standard (C l3:0) at a concentration of 4,000 ppm. The free fatty acid concentration was expressed as milligrams of fatty acid per 100 g of fat.
Decarboxylase activity. Decarboxylase activity was initially tested with an agar plate method as described by Lorenzo et al. (28). The plates were incubated at 37°C and examined after 72 h of incubation. A purple halo around a colony indicated amine production. The strains positive for decarboxylase activity with the agar plate method were further studied by inoculating 0.1 ml of cell suspension into 5 ml of the culture medium described by Joosten and Northolt (25) without agar or bromocresol purple and supplemented with ornithine (2%) or lysine (2%). After incubation at 37°C for 72 h with shaking, the biogenic amines in the culture medium were quantified by high-performance liquid chromatog raphy methods (28). The quantity of each biogenic amine was expressed in parts per million. Statistical analysis. All statistical analyses were performed using the computer program Statistica 8.0 for Windows (StatSoft Inc., Tulsa, OK). Significant differences were determined based on a one-way analysis of variance. Duncan’s test was used to identify significant differences among strains. Differences were considered significant at P < 0.05. RESULTS AND DISCUSSION
Table 2 shows the growth and metabolic characteristics of the Bacillus strains studied. The temperature and pH values for growth chosen for the assays were those more frequently found during sausage ripening. Because of the variability in the NaCl concentrations in sausages, high
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(a)
(c)
A
B
A
C
B
C
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O
M
D
M
kDa
FIGURE 1. SDS-PAGE findings fo r protein hydrolysis by Bacil lus strains, (a) Sarcoplasmic proteins. Lane A, purified sarcoplas mic proteins (GPI, glucose phosphate isomerase; E, enolase; CK, creatine phosphate kinase; A, aldolase; GH, glyceraldehyde phosphate dehydrogenase). Lane B, uninoculated control after 72 h o f incubation. Lanes C and D, samples containing different isolates after 72 h o f incubation: lane C, SA50; lane D, SBOl.Lane M, molecular weight standard, (b) Actin. Lane A, purified actin preparation (D, desmin; Act, actin; Tt, troponin T; Tr, tropomy osin; MLC, myosin light chain; Ti, troponin I). Lanes B and C, samples containing different isolates after 72 h o f incubation: lane B, SA06; lane C, SA26. Lane M, molecular weight standard, (c) Myosin. Lane A, purified myosin preparation (MHC, myosin heavy chain; ct-act, alpha-actinin; D, desmin; Act, actin; Tt, troponin T; Tr, tropomyosin; MLC, myosin light chain; Tc, troponin C). Lanes B through D, samples containing different isolates after 72 h o f incubation: lane B, SB17; lane C, SA39; lane D, SB15. Lane M, molecular weight standard.
concentrations of NaCl were tested with the assumption that strains able to grow under conditions of high NaCl concentrations would also be able to grow in lower NaCl concentrations. Of the 19 tested strains, only 9 B. subtilis and 1 B. amyloliquefaciens were able to grow at 10°C. However, all strains grew in the presence of 10% salt and at pH values of 5.0 and 5.5, which indicates high tolerance to salt and adaptation to acidic pH. Similar results have been reported by other authors (5, 9) for other Bacillus strains from different sources. Regarding the proteolytic activity based on the agar plate method, the number of strains for which plate wells had halos greater than or less than 25 mm in diameter is indicated in Table 2. The proteolytic activity of Bacillus isolates from traditional fermented sausages, cereals, and legume-based foods has been reported by numerous authors (6, 9, 23, 32, 35). In the present study, 100% of the strains hydrolyzed sarcoplasmic proteins, 94.7% hydrolyzed myo fibrillar proteins (Table 2). These results are in agreement with those obtained by Baruzzi et al. (3), who found high proteolytic activity in various species of Bacillus isolated from Italian sausages and concluded that Bacillus strains contributed to the development of textural and organoleptic characteristics of the sausages. Strains with proteolytic activity as indicated by results of the agar plate method were subsequently tested using electrophoresis techniques. Figure 1 shows the hydrolysis of sarcoplasmic protein (Fig. la), actin (Fig. lb), and myosin (Fig. lc) extracts by the Bacillus strains, the protein standard used for the protein fraction identification, and the purified protein extracts used as substrates in the assays. To our knowledge, this study is the first in which the proteolytic activity of Bacillus strains isolated from meat products has been characterized by SDS-PAGE. The sarcoplasmic protein extract used (Fig. la, lane A) contained several proteins of various sizes: approximately 102, 66, 60, 55 (glucose phosphate isomerase), 48 (enolase), 45 (creatine phosphate kinase), 42 (aldolase), 37 (glyceral dehyde phosphate dehydrogenase), 35, 25, and 24 kDa. Analysis of control samples reflected no changes in the protein profile (Fig. la, lane B). Of the 19 strains tested, 6 B. subtilis (SA50, SB07, SB 14, SB 17, SB 18, and SB26) and 4 B. amyloliquefaciens (SA35, SA37, SA39, and SA43) hydrolyzed all proteins present in the sarcoplasmic protein extract, resulting in complete absence of all the bands on the gel (Fig. la, lane C) and the generation of new peptides (at about 52, 44, 40, 30, 27, and < 20 kDa) in some cases. Strain SA06 (B. subtilis) hydrolyzed all the proteins except the protein of about 37 kDa (glyceraldehyde phosphate dehydrogenase) (data not shown). Strains SB01 and SB 13 (B. subtilis) hydrolyzed all proteins present in the extract except aldolase (42 kDa) (Fig. la, lane D). The remaining strains had lower proteolytic activity against the sarcoplas mic proteins (images not shown). For the actin extract, an intense band appeared at approximately 45 kDa (actin), and other bands appeared for many other polypeptides at 66, 55 (desmin), 37 (troponin T), 35 (tropomyosin), 25 (myosin light chain), and 24 (troponin T) kDa (Fig. lb, lane A). The strains studied had
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TABLE 3. Free fatty acid profiles of low, intermediate, and high lipolytic Bacillus strains Free fatty acid concn (mg/100 g of fat) Low lipolytic activity (n = 14)c
Intermediate lipolytic activity (n = 3)d
High lipolytic activity
Fatty acid(s)“
Mean
SEM
Mean
SEM
SB07
SB13
Significance6
C10:0 C12:0 0 4 :0 0 5 :0 0 6 :0 0 6 :1 0 7 :0 0 7 :1 0 8 :0 0 8 :1 n9 0 8 : 2 n6 0 8 : 3 n3 C20:0 C20:l n9 C20:2 n6 C20:4 n6 C22:0 C22:2 n6 C24:0 SFA UFA MUFA PUFA
13.25 24.52 22.92 9.29 69.85 22.94 9.47 11.63 44.01 77.84 38.17 19.37 19.60 12.09 11.64 11.78 5.17 7.63 12.60 236.62 207.14 124.50 82.64
3.20 1.95 0.59 1.07 6.42 1.75 0.43 0.30 3.70 8.11 4.17 1.30 0.39 0.37 0.98 0.26 2.28 1.60 3.05 14.00 14.16 8.81 5.83
8.56 27.16 25.96 11.08 141.64 21.52 15.87 13.89 114.39 205.20 87.38 15.81 21.32 16.47 16.45 12.83 9.66 12.32 22.36 397.99 401.87 257.09 144.78
8.56 0.69 1.59 0.33 25.85 2.80 1.29 0.58 14.63 21.75 11.50 1.19 0.34 0.70 0.72 0.60 5.64 0.27 0.54 38.10 27.54 20.65 13.66
26.03 29.18 57.59 13.26 603.34 47.71 38.72 20.56 295.50 410.29 167.00 23.35 23.04 20.02 19.25 12.52 19.98 15.92 26.03 1,131.70 736.63 498.58 238.05
27.95 32.04 70.85 15.26 730.12 53.43 45.52 21.65 374.25 450.23 208.59 25.37 26.68 22.28 21.24 13.93 21.54 19.78 32.11 1,376.32 836.50 547.59 288.91
NS NS ***
2,212.82
***
Total
443.76
26.70
799.86
61.07
1,868.33
NS *** *** *** ***
*** *** NS ** *
NS NS NS NS *** *** *** ***
a SFA, sum of saturated fatty acids; UFA, sum of unsaturated fatty acids; MUFA, sum of monounsaturated fatty acids; PUFA, sum of polyunsaturated fatty acids. * Significance of the interaction of low, intermediate, and high lipolytic activity: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, no significant difference. c Strains SA06, SA26, SA28, SA35, SA37, SA39, SA43, SA50, SB01, SB05, SB09, SB17, SB18, and SB26. d Strains SB 14, SB 15, and SB 16.
low activity against actin. Only one strain of B. amyloliquefaciens (SA26) had an actin band with reduced intensity (Fig. lb, lane C). No proteolytic changes were observed for the rest of the strains (Fig. lb, lane B) or in the uninoculated control. For the myosin extracts, an intense band appeared at approximately 200 kDa (myosin heavy chain), and other bands appeared for many other proteins at 102 (alphaactinin), 55 (desmin), 45 (actin), 37 (troponin T), 35 (tropomyosin), 25 (myosin light chain), 20 (troponin C), and 12 kDa (Fig. lc, lane A). Of the 19 strains tested, only 2 strains of B. suhtilis (SB 17 and SB26) did not have proteolytic activity against the myosin (Fig lc, lane B). Four strains of B. subtilis (SA06, SA50, SB 14, and SB 18) and two of B. amyloliquefaciens (SA39 and SA43) totally hydrolyzed the myosin heavy chain (Fig. lc, lane C). The actions of strains SA28, SB07, and SB 16 (B . subtilis) and SA35 (B . amyloliquefaciens) resulted in total hydrolysis of the myosin heavy chain and the generation of new peptides (at about 50, 46, and 42 kDa) (data not shown), whereas for the remaining strains the intensity of this protein band was reduced (Fig. lc, lane D). These findings cannot be compared with those of other studies because no data have
been previously reported regarding the proteolytic activity of Bacillus strains isolated from meat products as deter mined by SDS-PAGE techniques nor regarding the separated protein fractions (sarcoplasmic proteins, actin, and myosin). Concerning lipolytic activity, the titration method (Table 1) revealed that only two strains of B. subtilis had significant lipolytic activity (13.86 and 21.19% of oleic acid, respectively). These results are not totally in agreement with those reported by Baruzzi et al. (3) in Bacillus strains isolated from Italian sausages and by Ouoba et al. (33) in Bacillus strains isolated from a fermented African locust bean condiment, which indicates that lipolytic activity depends on the strain studied and its source. The strains with the highest lipolytic activity also had the highest values for total free fatty acids released, resulting in a significant positive correlation (r = 0.92; P < 0.001) between values of percentage of oleic acid and total free fatty acid concentrations. Table 3 shows the concentrations of the various fatty acids freed after 72 h of incubation quantified using gas chromatography techniques. Strains were grouped accord ing to their lipolytic activity: low (1,500 mg/100 g of fat). When comparing low versus intermediate versus high activity strains, significant differ ences (P < 0.001) were found regarding the sum of saturated fatty acids, sum of monounsaturated fatty acids, sum of polyunsaturated fatty acids, and total fatty acids freed. For strains SB07 and SB 13 with high lipolytic activity, the total free fatty acids were 1,868.33 and 2,212.82 mg/100 g of fat, respectively, and the saturated fatty acids were the main fraction, accounting for more than 60% of the total free fatty acids. Palmitic acid (C16:0) was the main fatty acid (32%), followed by oleic (C l8:1 n9), stearic (C l8:0), and linoleic (C l8:2 n6) acids. In the remaining strains, with intermediate or low lipolytic activity, the percentages for the saturated fatty acids and unsaturated fatty acids were similar, and the monounsatu rated fatty acids were predominant within the unsaturated fatty acids. These strains had similar profiles for free fatty acids; oleic acid was the main fatty acid followed by palmitic, stearic, and linoleic acids. These Bacillus strains differed in both their lipolytic activity (amount of fatty acid freed) and the kind of free fatty acids released. Triglycerides in pork subcutaneous fat have a characteristic distribution of fatty acid molecules. Most of the stearic acid (about 60%) is esterifying the sn 1 position of the glycerol, and most of the palmitic acid (60 to 80%) is esterifying the sn2 position; most of the oleic and linoleic acids (50 to 60%) are in position sn3 and less than 30% are in position snl (2, 7, 22). The free fatty acid profiles in these Bacillus strains reflect the specificity of their lipases for the three positions of the triglyceride molecule. Regarding decarboxylase activity, which was tested with agar plate method (Table 2), of the 19 Bacillus strains tested, 73.7% decarboxylated histidine, 94.7% decarboxylated tyrosine, and 73.7% decarboxylated ornithine and lysine, indicating the potential for these strains to produce biogenic amines. The most prevalent decarboxylase activity was found against tyrosine: 92.3% of B. subtilis strains and 100% of B. amyloliquefaciens strains decarboxylated this amino acid. Formation of biogenic amines in foods is important for consumer health and can result in off-flavors in foods (39). Biogenic amines may appear during sausage fermentation as a result of the bacterial decarboxylation reactions from precursor amino acids (19). The decarboxylase activity of Bacillus strains isolated from sausages has not been widely studied. Roig-Sagues et al. (37) analyzed four strains of Bacillus isolated from salchichon, a Spanish traditional sausage, and found that some strains had histidine decarboxylase activity. Histidine decarboxylase activity was also observed in several Bacillus isolates from salted semipreserved anchovies (21, 36), in agreement with the results of the present study. The amounts of putrescine and cadaverine formed by the Bacillus strains (Table 1) were generally low. In the B. subtilis strains, the accumulations were 0.39 to 18.43 ppm for putrescine and 0.43 to 4.29 ppm for cadaverine; in B. amyloliquefaciens strains, the accumulations were 0.76 to
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3.27 ppm for putrescine and 0.53 to 3.07 ppm for cadaverine. No data regarding the amount of biogenic amines produced by Bacillus strains of food origin have been published. Results of the present work indicate that amino acid decarboxylase activity is not particularly high in Bacillus strains, especially when compared with other microbial groups such as lactic acid bacteria or Enterobacteriaceae present in fermented meat products (4, 28). In the present study, strains of B. subtilis and B. amyloliquefaciens grew under the salt and pH environmen tal conditions typically found in sausages during ripening; however, only 9 of the 19 strains were able to grow at low temperatures. All tested strains had proteolytic activity against sarcoplasmic proteins and myosin, and two strains of B. subtilis also had significant lipolytic activity. These findings indicate that Bacillus strains may play an important role in the ripening of these sausages and in the development of their sensory properties. Among the strains characterized in the present study, B. amyloliquefaciens SA35 (isolated from Androlla) and B. subtilis SB07 (isolated from Botillo) grew under the environmental conditions tested, hydrolyzed sarcoplasmic proteins and myosin, and had lipolytic activity (very high activity in SB07 and significant activity in SA35). Both strains had low levels of decarboxylase activity. In future work, these two strains will be inoculated into experimental sausages to study their effects on the physicochemical, microbiological, and sensory characteristics of the final product, thus helping to elucidate definitively the role of these Bacillus strains in the ripening process of fermented sausages. ACKNOWLEDGMENTS This work was financially supported by the Xunta de Galicia (Regional Government) (project 07TAL021383PR). S. Fonseca acknowl edges financial support from the Spanish Ministry of Science and Innovation through a predoctoral FPU fellowship (AP2008-03385). The authors also thank the University of Vigo for financial support (Contracts Program with Reference Research Groups, call 2009, reference 09VIA12).
REFERENCES 1.
Abdel-Mohsen, H. A., M. Nakamoto, J. Kim, Y. Nogusa, K. Gekko, M. Ishioroshi, K. Samejima, S. Tanabe, and T. Nishimura. 2003. Changes in the properties of porcine myosin during postmortem aging. Food Sci. Technol. Res. 9:297-303. 2. Alford, J. A., J. L. Smith, and H. D. Lilly. 1971. Relationships of microbial activity to changes in lipids of foods. J. Appl. Bacteriol. 34: 133-146. 3. Baruzzi, F., A. Matarante, L. Caputo, and M. Morea. 2006. Molecular and physiological characterization of natural microbial communities isolated from a traditional Southern Italian processed sausage. Meat Sci. 72:261-269. 4. Bover-Cid, S., M. Hugas, M. Izquierdo-Pulido, and M. C. VidalCarou. 2001. Amino acid-decarboxylase activity of bacteria isolated from fermented pork sausages. Int. J. Food Microbiol. 66:185-189. 5. Calvo, P., and D. Zuniga. 2010. Caracterizacion fisiologica de cepas de Bacillus spp. aisladas de la rizosfera de papa (Solarium tuberosum). Ecol. Apl. 9:31-39. 6. Chantawannakul, P., A. Oncharoen, K. Klanbul, E. Chukeatirote, and S. Lumyong. 2002. Characterization of proteases of Bacillus subtilis strain 38 isolated from traditionally fermented soybean in northern Thailand. Sci. Asia 28:241-245.
J. Food Prot., Vol. 77, No. 9
7.
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Christie, W. W., and J. H. Moore. 1970. A comparison of the structure of triglycerides from various pig tissues. Biochim. Biophys. Acta 210:46-56. 8. Corbiere Morot-Bizot, S., S. Leroy, and R. Talon. 2006. Staphylo coccal community of a small unit manufacturing traditional dry fermented sausages. Int. J. Food Microbiol. 108:210-217. 9. Enemas, J., J. Sanz-Gomez, M. L. Garcla-Lopez, M. R. GarclaArmesto, and A. Otero. 1996. Evaluation of different systems for the identification of Bacillus strains isolated from Spanish fermented sausages. Meat Sci. 42:127-131. 10. Fadda, S„ Y. Sanz, G. Vignolo. M. Aristoy, G. Oliver, and F. Toldra. 1999. Characterization of muscle sarcoplasmic and myofibrillar protein hydrolysis caused by Lactobacillus plantarum. Appl. Environ. Microbiol. 65:3540-3546. 11. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. 12. Fonseca, S., A. Cachaldora, and J. Carballo. 2013. Characterization of actin and myosin extracts obtained using two improved laboratory methods. Food Anal. Methods 6:1033-1039. 13. Fonseca, S., A. Cachaldora, M. G6mez, I. Franco, and J. Carballo. 2013. Monitoring the bacterial population dynamics during the ripening of Galician chorizo, a traditional dry fermented Spanish sausage. Food Microbiol. 33:77—84. 14. Fonseca, S., L. I. I. Ouoba, I. Franco, and J. Carballo. 2013. Use of molecular methods to characterize the bacterial community and to monitor different native starter cultures throughout the ripening of chorizo gallego. Food Microbiol. 34:215—226. 15. Franco, I., M. C. Escamilla, J. Garcia, M. C. Garcia Fontan, and J. Carballo. 2006. Fatty acid profile of the fat from Celta pig breed fattened using a traditional feed: effect of the location in the carcass. J. Food Compos. Anal. 19:792-799. 16. Garcia-Fontan, M. C., J. M. Lorenzo, S. Martinez, I. Franco, and J. Carballo. 2007. Microbiological characteristics of Botillo, a Spanish traditional pork sausage. LWT Food Sci. Technol. 40:1610—1622. 17. Garcia-Fontan, M. C., J. M. Lorenzo, A. Parada, I. Franco, and J. Carballo. 2007. Microbiological characteristics of “ Androlla” , a Spanish traditional pork sausage. Food Microbiol. 24:52-58. 18. Garcia-Varona, M., E. M. Santos, I. Jaime, and J. Rovira. 2000. Characterisation of Micrococcaceae isolated from different varieties of chorizo. Int. J. Food Microbiol. 54:189-195. 19. Halasz, A., A. Barath, L. Simon-Sarkadi, and W. Holzapfel. 1994. Biogenic amines and their production by microorganisms in food. Trends Food Sci. Technol. 5:42-49. 20. Hechelmann, H., and R. Kasprowiak. 1991. Microbiological criteria for stable products. Fleischwirtschaft 71:1303-1307. 21. Hemandez-Herrero, M. M., A. X. Roig-Sagues, J. J. Rodrfguez-Jerez, and M. T. Mora-Ventura. 1999. Halotolerant and halophilic histamine-forming bacteria isolated during the ripening of salted anchovies (Engraulis encrasicholus). J. Food Prot. 62:509-514. 22. Hierro, E., L. de la Hoz, and J. A. Ordonez. 1997. Contribution of microbial and meat endogenous enzymes to the lipolysis of dryfermented sausages. J. Agric. Food Chem. 45:2989-2995. 23. Inatsu, Y., N. Nakamura, Y. Yuriko, T. Fushimi, L. Watanasiritum, and S. Kawamoto. 2006. Characterization of Bacillus subtilis in Thua Nao, a traditional fermented soybean food in northern Thailand. Lett. Appl. Microbiol. 43:237-242. 24. Iurlina, M. O., A. I. Saiz, S. R. Fuselli, and R. Fritz. 2006. Prevalence of Bacillus spp. in different food products collected in Argentina. LWT Food Sci. Technol. 39:105-110. 25. Joosten, H. M. L. J., and M. D. Northolt. 1989. Detection, growth, and amine-producing capacity of lactobacilli in cheese. Appl. Environ. Microbiol. 55:2356-2359.
26.
27. 28.
29.
30.
31.
32.
Kaluzny, M. A., L. A. Duncan, M. V. Merritt, and D. E, Epps. 1985. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J. Lipid Res. 26:135-140. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Lorenzo, J. M., A. Cachaldora, S. Fonseca, M. Gomez, I. Franco, and J. Carballo. 2010. Production of biogenic amines “ in vitro” in relation to the growth phase by Enterobacteriaceae species isolated from traditional sausages. Meat Sci. 86:684—691. Lorenzo, J. M„ M. Michinel, M. Lopez, and J. Carballo. 2000. Biochemical characteristics of two Spanish traditional dry-cured sausage varieties: Androlla and Botillo. J. Food Compos. Anal. 13: 809-817. Matarante, A., F. Baruzzi, P. S. Cocconcelli, and M. Morea. 2004. Genotyping and toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages. Appl. Environ. Microbiol. 70:5168-5176. Mauriello, G„ A. Casaburi, G. Blaiotta, and F. Villani. 2004. Isolation and technological properties of coagulase negative staph ylococci from fermented sausages of southern Italy. Meat Sci. 67: 149-158. Oguntoyinbo, F. A.; A. I. Sanni, C. M. Franz, and W. H. Holzapfel. 2007. In vitro fermentation studies for selection and evaluation of Bacillus strains as starter cultures for the production of okpehe, a traditional African fermented condiment. Int. J. Food Microbiol. 113: 208 218 Ouoba, L. I. I., M. D. Cantor, B. Diawara, A. S. Traore, and M. Jakobsen. 2003. Degradation of African locust bean oil by Bacillus subtilis and Bacillus pumilus isolated from soumbala, a fermented African locust bean condiment. J. Appl. Microbiol. 95:868—873. Perez-Juan, M., M. Flores, and F. Toldra. 2007. Simultaneous process to isolate actomyosin and actin from post-rigor porcine skeletal muscle. Food Chem. 101:1005-1011. Phromraksa, P., H. Nagano, T. Boonmars, and C. Kamboonruang. 2008. Identification of proteolytic bacteria from Thai traditional fermented foods and their allergenic reducing potentials. J. Food Sci. 73:M189-M195. Rodriguez-Jerez, J. J., M. T. Mora-Ventura, E. I. Lopez-Sabater, and M. Hemandez-Herrero. 1994. Histidine, lysine and ornithine decarboxylase bacteria in Spanish salted semi-preserved anchovies. J. Food Prot. 57:784-787. Roig-Sagues, A. X., M. Hemandez-Herrero, E. I. L6pez-Sabater, J. J. Rodriguez-Jerez, and M. T. Mora-Ventura. 1996. Histidine decar boxylase activity of bacteria isolated from raw and ripened salchichon, a Spanish cured sausage. J. Food Prot. 59:516-520. Shehata, A. J., J. M. De Man, and J. C. Alexander. 1970. A simple and rapid method for the preparation of methyl esters of fats in milligram amounts for gas chromatography. Can. Inst. Food Sci. Technol. J. 3:85-89. Suzzi, G„ and F. Gardini. 2003. Biogenic amines in dry fermented sausages: a review. Int. J. Food Microbiol. 88:41—54. Talon, R., S. Leroy, and I. Lebert. 2007. Microbial ecosystems of traditional fermented meat products: the importance of indigenous starters. Meat Sci. 77:55-62. Talon, R., D. Walter, S. Chattier, C. Barriere, and M. C. Montel. 1999. Effect of nitrate and incubation conditions on the production of catalase and nitrate reductase by staphylococci. Int. J. Food Microbiol. 52:47-56. Te Giffel, M. C., R. R. Beumer, S. Leijendekkers, and F. M. Rombouts. 1996. Incidence of Bacillus cereus and Bacillus subtilis in foods in the Netherlands. Food Microbiol. 13:53-58. Vignolo, G. M., A. P. Ruiz Holgado, and G. Oliver. 1988. Acid production and proteolytic activity of Lactobacillus strains isolated from dry sausages. J. Food Prot. 51:481^-84. -
33.
34.
35.
36.
37.
38.
39. 40.
41.
42.
43.
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Research Note
Metabolic Characterization of Bacillus subtilis and Bacillus amyloliquefaciens Strains Isolated from Traditional Dry-Cured Sausages AIDA CACHALDORA, SONIA FONSECA, MARIA GOM EZ, INMACULADA FRANCO, AND JAVIER CARBALLO* Area de Tecnologia de los Alimentos, Facultad de Ciencias de Ourense, Universidad de Vigo, 32004 Ourense, Spain MS 14-145: Received 27 March 2014/Accepted 29 April 2014
ABSTRACT The aim of this study was to investigate the effect of pH, temperature, and NaCl on growth, proteolytic and lipolytic activities, and the ability to produce biogenic amines of 19 strains of Bacillus isolated from Androlla and Botillo (two Spanish traditional sausages) to elucidate the role of these bacteria in sausage manufacture. All strains grew in the presence of 10% salt and at pH values of 5.0 and 5.5, whereas only 9 strains grew at 10°C. Proteolytic activity was assessed by the agar plate method, which revealed that 100 and 94.7% of the strains were able to hydrolyze sarcoplasmic and myofibrillar proteins, respectively. These results were confirmed by electrophoretic assays. The titration method revealed that only two strains hydrolyzed pork fat to any extent, and the profiles of the fatty acids freed were different. Most strains produced biogenic amines, but the quantities were generally low.
Raw-fermented sausages are widely consumed meat products in which the microbial activity plays an important role during the manufacturing process. Microorganisms are responsible for the fermentation process and mainly responsible for the proteolytic and lipolytic changes that occur during the ripening of these products, which determines their sensorial characteristics (texture, aroma, and taste). The microbiota of traditional dry-cured sausages is composed of a wide variety of microorganisms from the raw materials and the environment (13, 16-18, 40). Two major groups of bacteria dominate most of the ripening time and play well-known roles: lactic acid bacteria and gram positive coagulase-negative cocci, mainly represented by Staphylococcus and Kocuria (8). Lactic acid bacteria guarantee the safety of the products through the production of antimicrobial compounds such as lactic acid and bacteriocins, and coagulase-negative cocci contribute to color stability and enhance sensory properties due to liberation and degradation of amino acids and fatty acids (41). However, other microbial groups such as enterobac teria, Bacillus, and molds and yeasts may also proliferate, but their specific roles are not well understood (20). The genus Bacillus consists of a diverse array of gram positive aerobic and facultative anaerobic spore-forming rods that can be isolated from a wide variety of sources. Many species are important as food-spoilage organisms and can contaminate fruits, vegetable products, milk and other * Author for correspondence. Tel: + 34-988-387052; Fax: + 34-988387001; E-mail: [email protected].
dairy products, spices, and meat and meat products; however, these bacteria also play an important role in production of fermented products because of their proteo lytic activity (24, 30, 42). Several Bacillus species (B. subtilis, B. amyloliquefa ciens, B. pumilus, B. circulans, and B. megaterium) have been isolated from traditional dry-cured sausages (3, 9, 30). The presence of Bacillus in fermented sausages seems to be important. In a recent study (14), Bacillus strains were the only microorganisms isolated on manitol salt agar from a fermented sausage (Galician chorizo) meat mix before stuffing, with counts of 4.99 log CFU/g. Bacillus in fermented sausages have proteolytic and lipolytic activities (3), which could complement the effects of autochthonous enzymes from meat, lactic acid bacteria enzymes, and coagulase-negative cocci enzymes during sausage ripening. Therefore, the presence of Bacillus should have an effect (positive or negative) on the organoleptic characteristics of the final products. Androlla and Botillo are two traditional sausages widely made and consumed in northwestern Spain (29). Bacillus microorganisms have not been counted directly in Androlla or Botillo sausages. However, mean counts on standard plate count agar plus 7.5% NaCl were 6.87 log CFU/g for Androlla sausages (17) and 6.56 log CFU/g for Botillo sausages (76); approximately 10% of the bacterial strains from these sausages isolated on the standard plate count agar plus 7.5% NaCl culture medium were later identified as Bacillus species. The aim of this study was to determine the growth and metabolic properties of 19 strains of Bacillus isolated from
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TABLE 1. Bacillus strain identity, source sausage, lipolytic activity assessed by titration method, and biogenic amine productiona Biogenic amines (ppm) Strain no. SA06 SA26 SA28 SA35 SA37 SA39 SA43 SA50 SB01 SB05 SB07 SB09 SB13 SB14 SB15 SB16 SB17 SB 18 SB26
Species
B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B. B.
subtilis amyloliquefaciens subtilis amyloliquefaciens amyloliquefaciens amyloliquefaciens amyloliquefaciens subtilis subtilis subtilis subtilis amyloliquefaciens subtilis subtilis subtilis subtilis subtilis subtilis subtilis
Source Androlla Androlla Androlla Androlla Androlla Androlla Androlla Androlla Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo Botillo
Lipolytic activity (% oleic acid) 0.22 0.94 1.26 1.17 0.76 1.08 1.34 0.15 0.04 0.45 13.86 0.15 21.19 0.26 1.64 1.03 0.90 0.47 0.31
(0.20-0.24) (0.92-0.96) (1.23-1.29) (1.14-1.20) (0.74-0.78) (1.06-1.10) (1.30-1.38) (0.12-0.18) (0.02-0.06) (0.10-0.80) (13.58-14.14) (0.12-0.18) (21.03-21.35) (0.04-0.48) (1.61-1.69) (0.84-1.22) (0.85-0.95) (0.47-0.47) (0.29-0.33)
Putrescine 18.43 1.22 0.70 0.76 2.49 2.27 3.27 1.19 1.27 0.57 0.39 1.33 0.86
(16.80-20.03) (1.22-1.22) (0.60-0.80) (0.68-0.84) (2.46-2.52) (2.25-2.29) (3.15-3.39) (1.19-1.19) (1.27-1.27) (0.56-0.58) (0.38-0.40) (1.16-1.50) (0.61-1.11) ND ND ND ND ND 1.75 (1.54-1.96)
Cadaverine 0.43 3.07 0.44 1.08 0.53 0.56 2.52 4.29 3.08 0.58 0.59 0.62 0.52
(0.41-0.45) (3.07-3.07) (0.31-0.57) (0.76-1.40) (0.34-0.72) (0.29-0.83) (2.51-2.53) (4.29-4.29) (3.08-3.08) (0.51-0.65) (0.32-0.86) (0.54-0.70) (0.39-0.65) ND ND ND ND ND 0.50 (0.44-0.56)
" Values are means (ranges) of two replicates. ND, not detected.
Androlla and Botillo sausages. This is the first step in selecting appropriate strains for further studies (inoculation of experimental sausages and analysis of their physico chemical, microbiological, and organoleptic characteristics), determining the role of Bacillus spp. in the ripening of the fermented sausages, and understanding the effect of this group of microorganisms on the quality of the final products.
Effect of pH, temperature, and NaCl on microbial growth. Each strain was tested for its ability to grow at 10°C in BHI broth at pH 7.0, in BHI broth adjusted to pH 5 and 5.5 with lactic acid, and in BHI broth supplemented with 10 and 15% NaCl. Ten microliters of an overnight culture of each strain was inoculated into 5 ml of these test media, and the growth was scored as positive or negative after incubation at 37°C for 72 h. Growth was scored as positive when tubes were more turbid than the noninoculated and incubated tubes used as control.
MATERIALS AND METHODS
Proteolytic activity: qualitative assessment by the agar plate method. Sarcoplasmic and myofibrillar proteins were extracted according to the method of Fadda et al. (10). Sarcoplasmic proteins were sterilized by filtration through a polyvinylidene fluoride filter (0.22-pm pore size; Millipore, Billerica, MA). Myofibrillar proteins were extracted under sterile conditions. The sarcoplasmic and myofibrillar proteins were added at concentrations of 0.5 and 0.2 mg/ml, respectively, to sterile medium containing 0.25% yeast extract, 0.1% glucose, and 1.5% agar. The medium was poured into petri dishes, and after solidification, three wells were bored in the agar in each plate, and 40 pi of cell suspension was pipetted into each well. After incubation at 37°C for 48 h, the agar disc was removed from each dish and stained for 30 min in 0.05% (wt/vol) Coomassie blue R-250 (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in methanol-acetic acid-water (50:10:40) and destained in methanolethanol-acetic acid-water (20:10:5:65). The diameter of the clear zone surrounding each inoculated well was recorded as an indication of proteolytic activity.
Bacillus strains and preparation of cell suspensions. The B. subtilis and B. amyloliquefaciens strains used in this study (Table 1) were isolated on standard plate count agar (Oxoid, Basingstoke, UK) plus 7.5% NaCl (16, 17). For the Androlla and Botillo sausages, 13 of the 200 isolates and 17 of the 150 isolates, respectively, found on standard plate count agar plus 7.5% NaCl were later determined to be Bacillus spp. Strains were identified by sequencing the 16S rRNA gene and comparing the obtained sequences with those available in the GenBank database (National Center for Biotechnology Information, Bethesda, MD). The genomic DNA extracted from the Bacillus isolates was then subjected to repetitive sequence-based PCR analysis using the single oligonucleotide primer (GTG)5 as described by Fonseca et al. (14). The 19 strains selected for this study had different (GTG)5-PCR fingerprinting profiles, indicating differences among strains of the same species. Strains were stored at —80°C in brain heart infusion (BHI) broth (Oxoid) with 20% (vol/vol) glycerol as a cryoprotective agent. To prepare the cell suspensions, a correlation between the log CFU per milliliter and the absorbance at 650 nm was established for each strain. Samples of BHI broth cultures were collected after 24 h of incubation, and the absorbance at 650 nm was measured. The cultures were then centrifuged at 12,000 x g for 10 min, and the resulting pellets were washed twice with 20 mM phosphate buffer pH 7.0 and then resuspended in the same buffer to obtain inocula containing approximately 109 CFU/ml.
Proteolytic activity: quantitative assessment by electro phoretic methods. Strains that exhibited proteolytic activity by the agar plate method were then tested by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Actin and myosin were extracted and quantified using the methods described by Perez-Juan et al. (34) and Abdel-Mohsen et al. (1), respectively, with modifications in the centrifugation times and composition of
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TABLE 2. Bacillus strain growth and metabolic characteristics No. of positive isolates Characteristic
B. subtilis
B. amyloliquefaciens
13 13 9
6 6 1
13 10
6 3
13 (11, halo > 25 mm; 2, halo < 25 mm) 13 (11, halo > 25 mm; 2, halo < 25 mm)
6 (halo > 25 mm) 5 (4, halo < 25 mm; 1, halo < 25 mm)
10 12 8 8
4 6 6 6
13
6
Growth at: pH 5.0 pH 5.5 10°C Growth in NaCl 10% 15% Proteolytic activity0 Sarcoplasmic proteins Myofibrillar proteins Decarboxylase activity0 Histidine Tyrosine Ornithine Lysine Total no. of isolates tested ° Determined with the agar plate method. the buffers for resuspension (12). A 0.2-ml volume of cell suspension was inoculated with 1 ml of sarcoplasmic protein, actin, and myosin extract (0.15 mg/ml) supplemented with 1% glucose and incubated at 37°C for 72 h in a shaking incubator. Uninoculated protein extracts were used as controls and incubated under the same conditions. After incubation, samples were examined to determine proteolytic activity. Protein degradation was assayed by SDS-PAGE as described by Laemmli (27) on 12% polyacrylamide gels. Ten microliters of each protein preparation was mixed with 19 ml of Laemmli buffer (Bio-Rad, Hercules, CA) and 1 ml of [1-mcrcaptoethanol, and 30 pi of each preparation and 10 pi of SDS-PAGE molecular weight standard low range (BioRad) were placed in different wells in the gel. Electrophoresis was carried out at 220 V for about 45 min. Gels were soaked in a fixing solution of methanol-water-acetic acid (50:43:7) for 15 min, stained in 0.05% (wt/vol) Coomassie blue R-250 in methanolacetic acid-water (45:10:45) for 2 h, and destained until the background was clear. The molecular weights of the products of proteolysis were estimated by reference to the relative mobilities of standard proteins. The results corresponding to each band were expressed as a percentage of total absorbance at 550 nm calculated with Quantity One Software (Bio-Rad).
Lipolytic activity. Lipolytic activity was assessed using the method described by Vignolo et al. (43) with some modifications. One milliliter of a cell suspension of each strain was inoculated into 30 ml of a sterile broth containing 1% (wt/vol) peptone, 0.5% (wt/vol) yeast extract, 0.5% (wt/vol) meat extract, and 3% (wt/vol) NaCl, pH 7.0, supplemented with 15 g of pork fat. After incubation at 37°C for 72 h with shaking, the lipolytic activity was measured by titration. The fat was extracted according to the method of Folch et al. (11), and the free fatty acids were titrated following the procedure of Vignolo et al. (43). The free fatty acid concentration was expressed as percentage of oleic acid (31). The same medium was also used to determine the ability of the strains to release free fatty acids from triglycerides. After 72 h of incubation, the fat in each culture was extracted as described, and the free fatty acids were separated from the triglycerides in
columns of NH2-aminopropyl following the procedure described by Kaluzny et al. (26). Methyl esters of free fatty acids were prepared using the method of Shehata et al. (38) and were quantified by gas chromatography following the procedure described by Franco et al. (15) using an internal standard (C l3:0) at a concentration of 4,000 ppm. The free fatty acid concentration was expressed as milligrams of fatty acid per 100 g of fat.
Decarboxylase activity. Decarboxylase activity was initially tested with an agar plate method as described by Lorenzo et al. (28). The plates were incubated at 37°C and examined after 72 h of incubation. A purple halo around a colony indicated amine production. The strains positive for decarboxylase activity with the agar plate method were further studied by inoculating 0.1 ml of cell suspension into 5 ml of the culture medium described by Joosten and Northolt (25) without agar or bromocresol purple and supplemented with ornithine (2%) or lysine (2%). After incubation at 37°C for 72 h with shaking, the biogenic amines in the culture medium were quantified by high-performance liquid chromatog raphy methods (28). The quantity of each biogenic amine was expressed in parts per million. Statistical analysis. All statistical analyses were performed using the computer program Statistica 8.0 for Windows (StatSoft Inc., Tulsa, OK). Significant differences were determined based on a one-way analysis of variance. Duncan’s test was used to identify significant differences among strains. Differences were considered significant at P < 0.05. RESULTS AND DISCUSSION
Table 2 shows the growth and metabolic characteristics of the Bacillus strains studied. The temperature and pH values for growth chosen for the assays were those more frequently found during sausage ripening. Because of the variability in the NaCl concentrations in sausages, high
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(c)
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M
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M
kDa
FIGURE 1. SDS-PAGE findings fo r protein hydrolysis by Bacil lus strains, (a) Sarcoplasmic proteins. Lane A, purified sarcoplas mic proteins (GPI, glucose phosphate isomerase; E, enolase; CK, creatine phosphate kinase; A, aldolase; GH, glyceraldehyde phosphate dehydrogenase). Lane B, uninoculated control after 72 h o f incubation. Lanes C and D, samples containing different isolates after 72 h o f incubation: lane C, SA50; lane D, SBOl.Lane M, molecular weight standard, (b) Actin. Lane A, purified actin preparation (D, desmin; Act, actin; Tt, troponin T; Tr, tropomy osin; MLC, myosin light chain; Ti, troponin I). Lanes B and C, samples containing different isolates after 72 h o f incubation: lane B, SA06; lane C, SA26. Lane M, molecular weight standard, (c) Myosin. Lane A, purified myosin preparation (MHC, myosin heavy chain; ct-act, alpha-actinin; D, desmin; Act, actin; Tt, troponin T; Tr, tropomyosin; MLC, myosin light chain; Tc, troponin C). Lanes B through D, samples containing different isolates after 72 h o f incubation: lane B, SB17; lane C, SA39; lane D, SB15. Lane M, molecular weight standard.
concentrations of NaCl were tested with the assumption that strains able to grow under conditions of high NaCl concentrations would also be able to grow in lower NaCl concentrations. Of the 19 tested strains, only 9 B. subtilis and 1 B. amyloliquefaciens were able to grow at 10°C. However, all strains grew in the presence of 10% salt and at pH values of 5.0 and 5.5, which indicates high tolerance to salt and adaptation to acidic pH. Similar results have been reported by other authors (5, 9) for other Bacillus strains from different sources. Regarding the proteolytic activity based on the agar plate method, the number of strains for which plate wells had halos greater than or less than 25 mm in diameter is indicated in Table 2. The proteolytic activity of Bacillus isolates from traditional fermented sausages, cereals, and legume-based foods has been reported by numerous authors (6, 9, 23, 32, 35). In the present study, 100% of the strains hydrolyzed sarcoplasmic proteins, 94.7% hydrolyzed myo fibrillar proteins (Table 2). These results are in agreement with those obtained by Baruzzi et al. (3), who found high proteolytic activity in various species of Bacillus isolated from Italian sausages and concluded that Bacillus strains contributed to the development of textural and organoleptic characteristics of the sausages. Strains with proteolytic activity as indicated by results of the agar plate method were subsequently tested using electrophoresis techniques. Figure 1 shows the hydrolysis of sarcoplasmic protein (Fig. la), actin (Fig. lb), and myosin (Fig. lc) extracts by the Bacillus strains, the protein standard used for the protein fraction identification, and the purified protein extracts used as substrates in the assays. To our knowledge, this study is the first in which the proteolytic activity of Bacillus strains isolated from meat products has been characterized by SDS-PAGE. The sarcoplasmic protein extract used (Fig. la, lane A) contained several proteins of various sizes: approximately 102, 66, 60, 55 (glucose phosphate isomerase), 48 (enolase), 45 (creatine phosphate kinase), 42 (aldolase), 37 (glyceral dehyde phosphate dehydrogenase), 35, 25, and 24 kDa. Analysis of control samples reflected no changes in the protein profile (Fig. la, lane B). Of the 19 strains tested, 6 B. subtilis (SA50, SB07, SB 14, SB 17, SB 18, and SB26) and 4 B. amyloliquefaciens (SA35, SA37, SA39, and SA43) hydrolyzed all proteins present in the sarcoplasmic protein extract, resulting in complete absence of all the bands on the gel (Fig. la, lane C) and the generation of new peptides (at about 52, 44, 40, 30, 27, and < 20 kDa) in some cases. Strain SA06 (B. subtilis) hydrolyzed all the proteins except the protein of about 37 kDa (glyceraldehyde phosphate dehydrogenase) (data not shown). Strains SB01 and SB 13 (B. subtilis) hydrolyzed all proteins present in the extract except aldolase (42 kDa) (Fig. la, lane D). The remaining strains had lower proteolytic activity against the sarcoplas mic proteins (images not shown). For the actin extract, an intense band appeared at approximately 45 kDa (actin), and other bands appeared for many other polypeptides at 66, 55 (desmin), 37 (troponin T), 35 (tropomyosin), 25 (myosin light chain), and 24 (troponin T) kDa (Fig. lb, lane A). The strains studied had
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TABLE 3. Free fatty acid profiles of low, intermediate, and high lipolytic Bacillus strains Free fatty acid concn (mg/100 g of fat) Low lipolytic activity (n = 14)c
Intermediate lipolytic activity (n = 3)d
High lipolytic activity
Fatty acid(s)“
Mean
SEM
Mean
SEM
SB07
SB13
Significance6
C10:0 C12:0 0 4 :0 0 5 :0 0 6 :0 0 6 :1 0 7 :0 0 7 :1 0 8 :0 0 8 :1 n9 0 8 : 2 n6 0 8 : 3 n3 C20:0 C20:l n9 C20:2 n6 C20:4 n6 C22:0 C22:2 n6 C24:0 SFA UFA MUFA PUFA
13.25 24.52 22.92 9.29 69.85 22.94 9.47 11.63 44.01 77.84 38.17 19.37 19.60 12.09 11.64 11.78 5.17 7.63 12.60 236.62 207.14 124.50 82.64
3.20 1.95 0.59 1.07 6.42 1.75 0.43 0.30 3.70 8.11 4.17 1.30 0.39 0.37 0.98 0.26 2.28 1.60 3.05 14.00 14.16 8.81 5.83
8.56 27.16 25.96 11.08 141.64 21.52 15.87 13.89 114.39 205.20 87.38 15.81 21.32 16.47 16.45 12.83 9.66 12.32 22.36 397.99 401.87 257.09 144.78
8.56 0.69 1.59 0.33 25.85 2.80 1.29 0.58 14.63 21.75 11.50 1.19 0.34 0.70 0.72 0.60 5.64 0.27 0.54 38.10 27.54 20.65 13.66
26.03 29.18 57.59 13.26 603.34 47.71 38.72 20.56 295.50 410.29 167.00 23.35 23.04 20.02 19.25 12.52 19.98 15.92 26.03 1,131.70 736.63 498.58 238.05
27.95 32.04 70.85 15.26 730.12 53.43 45.52 21.65 374.25 450.23 208.59 25.37 26.68 22.28 21.24 13.93 21.54 19.78 32.11 1,376.32 836.50 547.59 288.91
NS NS ***
2,212.82
***
Total
443.76
26.70
799.86
61.07
1,868.33
NS *** *** *** ***
*** *** NS ** *
NS NS NS NS *** *** *** ***
a SFA, sum of saturated fatty acids; UFA, sum of unsaturated fatty acids; MUFA, sum of monounsaturated fatty acids; PUFA, sum of polyunsaturated fatty acids. * Significance of the interaction of low, intermediate, and high lipolytic activity: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, no significant difference. c Strains SA06, SA26, SA28, SA35, SA37, SA39, SA43, SA50, SB01, SB05, SB09, SB17, SB18, and SB26. d Strains SB 14, SB 15, and SB 16.
low activity against actin. Only one strain of B. amyloliquefaciens (SA26) had an actin band with reduced intensity (Fig. lb, lane C). No proteolytic changes were observed for the rest of the strains (Fig. lb, lane B) or in the uninoculated control. For the myosin extracts, an intense band appeared at approximately 200 kDa (myosin heavy chain), and other bands appeared for many other proteins at 102 (alphaactinin), 55 (desmin), 45 (actin), 37 (troponin T), 35 (tropomyosin), 25 (myosin light chain), 20 (troponin C), and 12 kDa (Fig. lc, lane A). Of the 19 strains tested, only 2 strains of B. suhtilis (SB 17 and SB26) did not have proteolytic activity against the myosin (Fig lc, lane B). Four strains of B. subtilis (SA06, SA50, SB 14, and SB 18) and two of B. amyloliquefaciens (SA39 and SA43) totally hydrolyzed the myosin heavy chain (Fig. lc, lane C). The actions of strains SA28, SB07, and SB 16 (B . subtilis) and SA35 (B . amyloliquefaciens) resulted in total hydrolysis of the myosin heavy chain and the generation of new peptides (at about 50, 46, and 42 kDa) (data not shown), whereas for the remaining strains the intensity of this protein band was reduced (Fig. lc, lane D). These findings cannot be compared with those of other studies because no data have
been previously reported regarding the proteolytic activity of Bacillus strains isolated from meat products as deter mined by SDS-PAGE techniques nor regarding the separated protein fractions (sarcoplasmic proteins, actin, and myosin). Concerning lipolytic activity, the titration method (Table 1) revealed that only two strains of B. subtilis had significant lipolytic activity (13.86 and 21.19% of oleic acid, respectively). These results are not totally in agreement with those reported by Baruzzi et al. (3) in Bacillus strains isolated from Italian sausages and by Ouoba et al. (33) in Bacillus strains isolated from a fermented African locust bean condiment, which indicates that lipolytic activity depends on the strain studied and its source. The strains with the highest lipolytic activity also had the highest values for total free fatty acids released, resulting in a significant positive correlation (r = 0.92; P < 0.001) between values of percentage of oleic acid and total free fatty acid concentrations. Table 3 shows the concentrations of the various fatty acids freed after 72 h of incubation quantified using gas chromatography techniques. Strains were grouped accord ing to their lipolytic activity: low (1,500 mg/100 g of fat). When comparing low versus intermediate versus high activity strains, significant differ ences (P < 0.001) were found regarding the sum of saturated fatty acids, sum of monounsaturated fatty acids, sum of polyunsaturated fatty acids, and total fatty acids freed. For strains SB07 and SB 13 with high lipolytic activity, the total free fatty acids were 1,868.33 and 2,212.82 mg/100 g of fat, respectively, and the saturated fatty acids were the main fraction, accounting for more than 60% of the total free fatty acids. Palmitic acid (C16:0) was the main fatty acid (32%), followed by oleic (C l8:1 n9), stearic (C l8:0), and linoleic (C l8:2 n6) acids. In the remaining strains, with intermediate or low lipolytic activity, the percentages for the saturated fatty acids and unsaturated fatty acids were similar, and the monounsatu rated fatty acids were predominant within the unsaturated fatty acids. These strains had similar profiles for free fatty acids; oleic acid was the main fatty acid followed by palmitic, stearic, and linoleic acids. These Bacillus strains differed in both their lipolytic activity (amount of fatty acid freed) and the kind of free fatty acids released. Triglycerides in pork subcutaneous fat have a characteristic distribution of fatty acid molecules. Most of the stearic acid (about 60%) is esterifying the sn 1 position of the glycerol, and most of the palmitic acid (60 to 80%) is esterifying the sn2 position; most of the oleic and linoleic acids (50 to 60%) are in position sn3 and less than 30% are in position snl (2, 7, 22). The free fatty acid profiles in these Bacillus strains reflect the specificity of their lipases for the three positions of the triglyceride molecule. Regarding decarboxylase activity, which was tested with agar plate method (Table 2), of the 19 Bacillus strains tested, 73.7% decarboxylated histidine, 94.7% decarboxylated tyrosine, and 73.7% decarboxylated ornithine and lysine, indicating the potential for these strains to produce biogenic amines. The most prevalent decarboxylase activity was found against tyrosine: 92.3% of B. subtilis strains and 100% of B. amyloliquefaciens strains decarboxylated this amino acid. Formation of biogenic amines in foods is important for consumer health and can result in off-flavors in foods (39). Biogenic amines may appear during sausage fermentation as a result of the bacterial decarboxylation reactions from precursor amino acids (19). The decarboxylase activity of Bacillus strains isolated from sausages has not been widely studied. Roig-Sagues et al. (37) analyzed four strains of Bacillus isolated from salchichon, a Spanish traditional sausage, and found that some strains had histidine decarboxylase activity. Histidine decarboxylase activity was also observed in several Bacillus isolates from salted semipreserved anchovies (21, 36), in agreement with the results of the present study. The amounts of putrescine and cadaverine formed by the Bacillus strains (Table 1) were generally low. In the B. subtilis strains, the accumulations were 0.39 to 18.43 ppm for putrescine and 0.43 to 4.29 ppm for cadaverine; in B. amyloliquefaciens strains, the accumulations were 0.76 to
J. Food Prot., Vol. 77, No. 9
3.27 ppm for putrescine and 0.53 to 3.07 ppm for cadaverine. No data regarding the amount of biogenic amines produced by Bacillus strains of food origin have been published. Results of the present work indicate that amino acid decarboxylase activity is not particularly high in Bacillus strains, especially when compared with other microbial groups such as lactic acid bacteria or Enterobacteriaceae present in fermented meat products (4, 28). In the present study, strains of B. subtilis and B. amyloliquefaciens grew under the salt and pH environmen tal conditions typically found in sausages during ripening; however, only 9 of the 19 strains were able to grow at low temperatures. All tested strains had proteolytic activity against sarcoplasmic proteins and myosin, and two strains of B. subtilis also had significant lipolytic activity. These findings indicate that Bacillus strains may play an important role in the ripening of these sausages and in the development of their sensory properties. Among the strains characterized in the present study, B. amyloliquefaciens SA35 (isolated from Androlla) and B. subtilis SB07 (isolated from Botillo) grew under the environmental conditions tested, hydrolyzed sarcoplasmic proteins and myosin, and had lipolytic activity (very high activity in SB07 and significant activity in SA35). Both strains had low levels of decarboxylase activity. In future work, these two strains will be inoculated into experimental sausages to study their effects on the physicochemical, microbiological, and sensory characteristics of the final product, thus helping to elucidate definitively the role of these Bacillus strains in the ripening process of fermented sausages. ACKNOWLEDGMENTS This work was financially supported by the Xunta de Galicia (Regional Government) (project 07TAL021383PR). S. Fonseca acknowl edges financial support from the Spanish Ministry of Science and Innovation through a predoctoral FPU fellowship (AP2008-03385). The authors also thank the University of Vigo for financial support (Contracts Program with Reference Research Groups, call 2009, reference 09VIA12).
REFERENCES 1.
Abdel-Mohsen, H. A., M. Nakamoto, J. Kim, Y. Nogusa, K. Gekko, M. Ishioroshi, K. Samejima, S. Tanabe, and T. Nishimura. 2003. Changes in the properties of porcine myosin during postmortem aging. Food Sci. Technol. Res. 9:297-303. 2. Alford, J. A., J. L. Smith, and H. D. Lilly. 1971. Relationships of microbial activity to changes in lipids of foods. J. Appl. Bacteriol. 34: 133-146. 3. Baruzzi, F., A. Matarante, L. Caputo, and M. Morea. 2006. Molecular and physiological characterization of natural microbial communities isolated from a traditional Southern Italian processed sausage. Meat Sci. 72:261-269. 4. Bover-Cid, S., M. Hugas, M. Izquierdo-Pulido, and M. C. VidalCarou. 2001. Amino acid-decarboxylase activity of bacteria isolated from fermented pork sausages. Int. J. Food Microbiol. 66:185-189. 5. Calvo, P., and D. Zuniga. 2010. Caracterizacion fisiologica de cepas de Bacillus spp. aisladas de la rizosfera de papa (Solarium tuberosum). Ecol. Apl. 9:31-39. 6. Chantawannakul, P., A. Oncharoen, K. Klanbul, E. Chukeatirote, and S. Lumyong. 2002. Characterization of proteases of Bacillus subtilis strain 38 isolated from traditionally fermented soybean in northern Thailand. Sci. Asia 28:241-245.
J. Food Prot., Vol. 77, No. 9
7.
METABOLIC STUDY OF BACILLUS STRAINS FROM SAUSAGES
Christie, W. W., and J. H. Moore. 1970. A comparison of the structure of triglycerides from various pig tissues. Biochim. Biophys. Acta 210:46-56. 8. Corbiere Morot-Bizot, S., S. Leroy, and R. Talon. 2006. Staphylo coccal community of a small unit manufacturing traditional dry fermented sausages. Int. J. Food Microbiol. 108:210-217. 9. Enemas, J., J. Sanz-Gomez, M. L. Garcla-Lopez, M. R. GarclaArmesto, and A. Otero. 1996. Evaluation of different systems for the identification of Bacillus strains isolated from Spanish fermented sausages. Meat Sci. 42:127-131. 10. Fadda, S„ Y. Sanz, G. Vignolo. M. Aristoy, G. Oliver, and F. Toldra. 1999. Characterization of muscle sarcoplasmic and myofibrillar protein hydrolysis caused by Lactobacillus plantarum. Appl. Environ. Microbiol. 65:3540-3546. 11. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. 12. Fonseca, S., A. Cachaldora, and J. Carballo. 2013. Characterization of actin and myosin extracts obtained using two improved laboratory methods. Food Anal. Methods 6:1033-1039. 13. Fonseca, S., A. Cachaldora, M. G6mez, I. Franco, and J. Carballo. 2013. Monitoring the bacterial population dynamics during the ripening of Galician chorizo, a traditional dry fermented Spanish sausage. Food Microbiol. 33:77—84. 14. Fonseca, S., L. I. I. Ouoba, I. Franco, and J. Carballo. 2013. Use of molecular methods to characterize the bacterial community and to monitor different native starter cultures throughout the ripening of chorizo gallego. Food Microbiol. 34:215—226. 15. Franco, I., M. C. Escamilla, J. Garcia, M. C. Garcia Fontan, and J. Carballo. 2006. Fatty acid profile of the fat from Celta pig breed fattened using a traditional feed: effect of the location in the carcass. J. Food Compos. Anal. 19:792-799. 16. Garcia-Fontan, M. C., J. M. Lorenzo, S. Martinez, I. Franco, and J. Carballo. 2007. Microbiological characteristics of Botillo, a Spanish traditional pork sausage. LWT Food Sci. Technol. 40:1610—1622. 17. Garcia-Fontan, M. C., J. M. Lorenzo, A. Parada, I. Franco, and J. Carballo. 2007. Microbiological characteristics of “ Androlla” , a Spanish traditional pork sausage. Food Microbiol. 24:52-58. 18. Garcia-Varona, M., E. M. Santos, I. Jaime, and J. Rovira. 2000. Characterisation of Micrococcaceae isolated from different varieties of chorizo. Int. J. Food Microbiol. 54:189-195. 19. Halasz, A., A. Barath, L. Simon-Sarkadi, and W. Holzapfel. 1994. Biogenic amines and their production by microorganisms in food. Trends Food Sci. Technol. 5:42-49. 20. Hechelmann, H., and R. Kasprowiak. 1991. Microbiological criteria for stable products. Fleischwirtschaft 71:1303-1307. 21. Hemandez-Herrero, M. M., A. X. Roig-Sagues, J. J. Rodrfguez-Jerez, and M. T. Mora-Ventura. 1999. Halotolerant and halophilic histamine-forming bacteria isolated during the ripening of salted anchovies (Engraulis encrasicholus). J. Food Prot. 62:509-514. 22. Hierro, E., L. de la Hoz, and J. A. Ordonez. 1997. Contribution of microbial and meat endogenous enzymes to the lipolysis of dryfermented sausages. J. Agric. Food Chem. 45:2989-2995. 23. Inatsu, Y., N. Nakamura, Y. Yuriko, T. Fushimi, L. Watanasiritum, and S. Kawamoto. 2006. Characterization of Bacillus subtilis in Thua Nao, a traditional fermented soybean food in northern Thailand. Lett. Appl. Microbiol. 43:237-242. 24. Iurlina, M. O., A. I. Saiz, S. R. Fuselli, and R. Fritz. 2006. Prevalence of Bacillus spp. in different food products collected in Argentina. LWT Food Sci. Technol. 39:105-110. 25. Joosten, H. M. L. J., and M. D. Northolt. 1989. Detection, growth, and amine-producing capacity of lactobacilli in cheese. Appl. Environ. Microbiol. 55:2356-2359.
26.
27. 28.
29.
30.
31.
32.
Kaluzny, M. A., L. A. Duncan, M. V. Merritt, and D. E, Epps. 1985. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J. Lipid Res. 26:135-140. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Lorenzo, J. M., A. Cachaldora, S. Fonseca, M. Gomez, I. Franco, and J. Carballo. 2010. Production of biogenic amines “ in vitro” in relation to the growth phase by Enterobacteriaceae species isolated from traditional sausages. Meat Sci. 86:684—691. Lorenzo, J. M„ M. Michinel, M. Lopez, and J. Carballo. 2000. Biochemical characteristics of two Spanish traditional dry-cured sausage varieties: Androlla and Botillo. J. Food Compos. Anal. 13: 809-817. Matarante, A., F. Baruzzi, P. S. Cocconcelli, and M. Morea. 2004. Genotyping and toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages. Appl. Environ. Microbiol. 70:5168-5176. Mauriello, G„ A. Casaburi, G. Blaiotta, and F. Villani. 2004. Isolation and technological properties of coagulase negative staph ylococci from fermented sausages of southern Italy. Meat Sci. 67: 149-158. Oguntoyinbo, F. A.; A. I. Sanni, C. M. Franz, and W. H. Holzapfel. 2007. In vitro fermentation studies for selection and evaluation of Bacillus strains as starter cultures for the production of okpehe, a traditional African fermented condiment. Int. J. Food Microbiol. 113: 208 218 Ouoba, L. I. I., M. D. Cantor, B. Diawara, A. S. Traore, and M. Jakobsen. 2003. Degradation of African locust bean oil by Bacillus subtilis and Bacillus pumilus isolated from soumbala, a fermented African locust bean condiment. J. Appl. Microbiol. 95:868—873. Perez-Juan, M., M. Flores, and F. Toldra. 2007. Simultaneous process to isolate actomyosin and actin from post-rigor porcine skeletal muscle. Food Chem. 101:1005-1011. Phromraksa, P., H. Nagano, T. Boonmars, and C. Kamboonruang. 2008. Identification of proteolytic bacteria from Thai traditional fermented foods and their allergenic reducing potentials. J. Food Sci. 73:M189-M195. Rodriguez-Jerez, J. J., M. T. Mora-Ventura, E. I. Lopez-Sabater, and M. Hemandez-Herrero. 1994. Histidine, lysine and ornithine decarboxylase bacteria in Spanish salted semi-preserved anchovies. J. Food Prot. 57:784-787. Roig-Sagues, A. X., M. Hemandez-Herrero, E. I. L6pez-Sabater, J. J. Rodriguez-Jerez, and M. T. Mora-Ventura. 1996. Histidine decar boxylase activity of bacteria isolated from raw and ripened salchichon, a Spanish cured sausage. J. Food Prot. 59:516-520. Shehata, A. J., J. M. De Man, and J. C. Alexander. 1970. A simple and rapid method for the preparation of methyl esters of fats in milligram amounts for gas chromatography. Can. Inst. Food Sci. Technol. J. 3:85-89. Suzzi, G„ and F. Gardini. 2003. Biogenic amines in dry fermented sausages: a review. Int. J. Food Microbiol. 88:41—54. Talon, R., S. Leroy, and I. Lebert. 2007. Microbial ecosystems of traditional fermented meat products: the importance of indigenous starters. Meat Sci. 77:55-62. Talon, R., D. Walter, S. Chattier, C. Barriere, and M. C. Montel. 1999. Effect of nitrate and incubation conditions on the production of catalase and nitrate reductase by staphylococci. Int. J. Food Microbiol. 52:47-56. Te Giffel, M. C., R. R. Beumer, S. Leijendekkers, and F. M. Rombouts. 1996. Incidence of Bacillus cereus and Bacillus subtilis in foods in the Netherlands. Food Microbiol. 13:53-58. Vignolo, G. M., A. P. Ruiz Holgado, and G. Oliver. 1988. Acid production and proteolytic activity of Lactobacillus strains isolated from dry sausages. J. Food Prot. 51:481^-84. -
33.
34.
35.
36.
37.
38.
39. 40.
41.
42.
43.
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.
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