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Journal of Food Protection, Vol. 77, No. 9, 2014, Pages 1605–1611 doi:10.4315/0362-028X.JFP-14-145 Copyright G, International Association for Food Protection

Research Note Metabolic Characterization of Bacillus subtilis and Bacillus amyloliquefaciens Strains Isolated from Traditional Dry-Cured

AIDA CACHALDORA, SONIA FONSECA, MARI´AGO´ MEZ, INMACULADA FRANCO, AND JAVIER CARBALLO* Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/9/1605/1687240/0362-028x_jfp-14-145.pdf by guest on 26 September 2021 A´ rea de Tecnologı´a 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 and (two Spanish traditional sausages) to elucidate the role of these bacteria in 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 10uC. 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 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 dairy products, , and meat and meat products; products in which the microbial activity plays an important however, these bacteria also play an important role in role during the manufacturing process. Microorganisms are production of fermented products because of their proteo- responsible for the fermentation process and mainly lytic activity (24, 30, 42). responsible for the proteolytic and lipolytic changes that Several Bacillus species (B. subtilis, B. amyloliquefa- occur during the ripening of these products, which ciens, B. pumilus, B. circulans, and B. megaterium) have determines their sensorial characteristics (texture, aroma, been isolated from traditional dry-cured sausages (3, 9, 30). and taste). The presence of Bacillus in fermented sausages seems to be The microbiota of traditional dry-cured sausages is important. In a recent study (14), Bacillus strains were the composed of a wide variety of microorganisms from the raw only microorganisms isolated on manitol salt agar from a materials and the environment (13, 16–18, 40). Two major (Galician ) meat mix before groups of bacteria dominate most of the ripening time and stuffing, with counts of 4.99 log CFU/g. Bacillus in play well-known roles: lactic acid bacteria and gram- fermented sausages have proteolytic and lipolytic activities positive coagulase-negative cocci, mainly represented by (3), which could complement the effects of autochthonous Staphylococcus and Kocuria (8). Lactic acid bacteria enzymes from meat, lactic acid bacteria enzymes, and guarantee the safety of the products through the production coagulase-negative cocci enzymes during sausage ripening. of antimicrobial compounds such as lactic acid and Therefore, the presence of Bacillus should have an effect bacteriocins, and coagulase-negative cocci contribute to (positive or negative) on the organoleptic characteristics of color stability and enhance sensory properties due to the final products. liberation and degradation of amino acids and fatty acids Androlla and Botillo are two traditional sausages (41). However, other microbial groups such as enterobac- widely made and consumed in northwestern Spain (29). teria, Bacillus, and molds and yeasts may also proliferate, Bacillus microorganisms have not been counted directly in but their specific roles are not well understood (20). Androlla or Botillo sausages. However, mean counts on The genus Bacillus consists of a diverse array of gram- standard plate count agar plus 7.5% NaCl were 6.87 log positive aerobic and facultative anaerobic spore-forming CFU/g for Androlla sausages (17) and 6.56 log CFU/g for rods that can be isolated from a wide variety of sources. Botillo sausages (16); approximately 10% of the bacterial Many species are important as food-spoilage organisms and strains from these sausages isolated on the standard plate can contaminate fruits, vegetable products, milk and other count agar plus 7.5% NaCl culture medium were later identified as Bacillus species. * Author for correspondence. Tel: z 34-988-387052; Fax: z 34-988- The aim of this study was to determine the growth and 387001; E-mail: [email protected]. metabolic properties of 19 strains of Bacillus isolated from 1606 CACHALDORA ET AL. J. Food Prot., Vol. 77, No. 9

TABLE 1. Bacillus strain identity, source sausage, lipolytic activity assessed by titration method, and biogenic amine productiona Biogenic amines (ppm) Lipolytic activity Strain no. Species Source (% oleic acid) Putrescine Cadaverine

SA06 B. subtilis Androlla 0.22 (0.20–0.24) 18.43 (16.80–20.03) 0.43 (0.41–0.45) SA26 B. amyloliquefaciens Androlla 0.94 (0.92–0.96) 1.22 (1.22–1.22) 3.07 (3.07–3.07) SA28 B. subtilis Androlla 1.26 (1.23–1.29) 0.70 (0.60–0.80) 0.44 (0.31–0.57) SA35 B. amyloliquefaciens Androlla 1.17 (1.14–1.20) 0.76 (0.68–0.84) 1.08 (0.76–1.40) SA37 B. amyloliquefaciens Androlla 0.76 (0.74–0.78) 2.49 (2.46–2.52) 0.53 (0.34–0.72) SA39 B. amyloliquefaciens Androlla 1.08 (1.06–1.10) 2.27 (2.25–2.29) 0.56 (0.29–0.83) SA43 B. amyloliquefaciens Androlla 1.34 (1.30–1.38) 3.27 (3.15–3.39) 2.52 (2.51–2.53) SA50 B. subtilis Androlla 0.15 (0.12–0.18) 1.19 (1.19–1.19) 4.29 (4.29–4.29) SB01 B. subtilis Botillo 0.04 (0.02–0.06) 1.27 (1.27–1.27) 3.08 (3.08–3.08) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/9/1605/1687240/0362-028x_jfp-14-145.pdf by guest on 26 September 2021 SB05 B. subtilis Botillo 0.45 (0.10–0.80) 0.57 (0.56–0.58) 0.58 (0.51–0.65) SB07 B. subtilis Botillo 13.86 (13.58–14.14) 0.39 (0.38–0.40) 0.59 (0.32–0.86) SB09 B. amyloliquefaciens Botillo 0.15 (0.12–0.18) 1.33 (1.16–1.50) 0.62 (0.54–0.70) SB13 B. subtilis Botillo 21.19 (21.03–21.35) 0.86 (0.61–1.11) 0.52 (0.39–0.65) SB14 B. subtilis Botillo 0.26 (0.04–0.48) ND ND SB15 B. subtilis Botillo 1.64 (1.61–1.69) ND ND SB16 B. subtilis Botillo 1.03 (0.84–1.22) ND ND SB17 B. subtilis Botillo 0.90 (0.85–0.95) ND ND SB18 B. subtilis Botillo 0.47 (0.47–0.47) ND ND SB26 B. subtilis Botillo 0.31 (0.29–0.33) 1.75 (1.54–1.96) 0.50 (0.44–0.56) a Values are means (ranges) of two replicates. ND, not detected.

Androlla and Botillo sausages. This is the first step in Effect of pH, temperature, and NaCl on microbial selecting appropriate strains for further studies (inoculation growth. Each strain was tested for its ability to grow at 10uCin of experimental sausages and analysis of their physico- BHI broth at pH 7.0, in BHI broth adjusted to pH 5 and 5.5 with chemical, microbiological, and organoleptic characteristics), lactic acid, and in BHI broth supplemented with 10 and 15% NaCl. determining the role of Bacillus spp. in the ripening of the Ten microliters of an overnight culture of each strain was inoculated into 5 ml of these test media, and the growth was fermented sausages, and understanding the effect of this scored as positive or negative after incubation at 37uC for 72 h. group of microorganisms on the quality of the final Growth was scored as positive when tubes were more turbid than products. the noninoculated and incubated tubes used as control.

MATERIALS AND METHODS Proteolytic activity: qualitative assessment by the agar Bacillus strains and preparation of cell suspensions. The plate method. Sarcoplasmic and myofibrillar proteins were B. subtilis and B. amyloliquefaciens strains used in this study extracted according to the method of Fadda et al. (10). (Table 1) were isolated on standard plate count agar (Oxoid, Sarcoplasmic proteins were sterilized by filtration through a Basingstoke, UK) plus 7.5% NaCl (16, 17). For the Androlla and polyvinylidene fluoride filter (0.22-mm pore size; Millipore, Botillo sausages, 13 of the 200 isolates and 17 of the 150 isolates, Billerica, MA). Myofibrillar proteins were extracted under sterile respectively, found on standard plate count agar plus 7.5% NaCl conditions. The sarcoplasmic and myofibrillar proteins were added were later determined to be Bacillus spp. Strains were identified by at concentrations of 0.5 and 0.2 mg/ml, respectively, to sterile sequencing the 16S rRNA gene and comparing the obtained medium containing 0.25% yeast extract, 0.1% glucose, and 1.5% sequences with those available in the GenBank database (National agar. The medium was poured into petri dishes, and after Center for Biotechnology Information, Bethesda, MD). The solidification, three wells were bored in the agar in each plate, genomic DNA extracted from the Bacillus isolates was then and 40 ml of cell suspension was pipetted into each well. After subjected to repetitive sequence–based PCR analysis using the incubation at 37uC for 48 h, the agar disc was removed from each single oligonucleotide primer (GTG)5 as described by Fonseca dish and stained for 30 min in 0.05% (wt/vol) Coomassie blue et al. (14). The 19 strains selected for this study had different R-250 (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) in (GTG)5-PCR fingerprinting profiles, indicating differences among methanol–acetic acid–water (50:10:40) and destained in methanol– strains of the same species. Strains were stored at 280uC in brain ethanol–acetic acid–water (20:10:5:65). The diameter of the clear heart infusion (BHI) broth (Oxoid) with 20% (vol/vol) glycerol as zone surrounding each inoculated well was recorded as an a cryoprotective agent. indication of proteolytic activity. To prepare the cell suspensions, a correlation between the log CFU per milliliter and the absorbance at 650 nm was established Proteolytic activity: quantitative assessment by electro- for each strain. Samples of BHI broth cultures were collected after phoretic methods. Strains that exhibited proteolytic activity by the 24 h of incubation, and the absorbance at 650 nm was measured. agar plate method were then tested by sodium dodecyl sulfate– The cultures were then centrifuged at 12,000 | g for 10 min, and polyacrylamide gel electrophoresis (SDS-PAGE). Actin and the resulting pellets were washed twice with 20 mM phosphate myosin were extracted and quantified using the methods described buffer pH 7.0 and then resuspended in the same buffer to obtain by Pe´rez-Juan et al. (34) and Abdel-Mohsen et al. (1), respectively, inocula containing approximately 109 CFU/ml. with modifications in the centrifugation times and composition of J. Food Prot., Vol. 77, No. 9 METABOLIC STUDY OF BACILLUS STRAINS FROM SAUSAGES 1607

TABLE 2. Bacillus strain growth and metabolic characteristics No. of positive isolates

Characteristic B. subtilis B. amyloliquefaciens

Growth at: pH 5.0 13 6 pH 5.5 13 6 10uC91 Growth in NaCl 10% 13 6 15% 10 3 Proteolytic activitya Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/9/1605/1687240/0362-028x_jfp-14-145.pdf by guest on 26 September 2021 Sarcoplasmic proteins 13 (11, halo . 25 mm; 2, halo , 25 mm) 6 (halo . 25 mm) Myofibrillar proteins 13 (11, halo . 25 mm; 2, halo , 25 mm) 5 (4, halo , 25 mm; 1, halo , 25 mm) Decarboxylase activitya Histidine 10 4 Tyrosine 12 6 Ornithine 8 6 Lysine 8 6 Total no. of isolates tested 13 6 a Determined with the agar plate method.

the buffers for resuspension (12). A 0.2-ml volume of cell columns of NH2-aminopropyl following the procedure described suspension was inoculated with 1 ml of sarcoplasmic protein, actin, by Kaluzny et al. (26). Methyl esters of free fatty acids were and myosin extract (0.15 mg/ml) supplemented with 1% glucose prepared using the method of Shehata et al. (38) and were and incubated at 37uC for 72 h in a shaking incubator. quantified by gas chromatography following the procedure Uninoculated protein extracts were used as controls and incubated described by Franco et al. (15) using an internal standard under the same conditions. After incubation, samples were (C13:0) at a concentration of 4,000 ppm. The free fatty acid examined to determine proteolytic activity. Protein degradation concentration was expressed as milligrams of fatty acid per 100 g was assayed by SDS-PAGE as described by Laemmli (27) on 12% of fat. polyacrylamide gels. Ten microliters of each protein preparation was mixed with 19 ml of Laemmli buffer (Bio-Rad, Hercules, CA) Decarboxylase activity. Decarboxylase activity was initially and 1 ml of b-mercaptoethanol, and 30 ml of each preparation and tested with an agar plate method as described by Lorenzo et al. 10 ml of SDS-PAGE molecular weight standard low range (Bio- (28). The plates were incubated at 37uC and examined after 72 h of Rad) were placed in different wells in the gel. Electrophoresis was incubation. A purple halo around a colony indicated amine carried out at 220 V for about 45 min. Gels were soaked in a fixing production. solution of methanol–water–acetic acid (50:43:7) for 15 min, The strains positive for decarboxylase activity with the agar stained in 0.05% (wt/vol) Coomassie blue R-250 in methanol– plate method were further studied by inoculating 0.1 ml of cell acetic acid–water (45:10:45) for 2 h, and destained until the suspension into 5 ml of the culture medium described by Joosten background was clear. The molecular weights of the products of and Northolt (25) without agar or bromocresol purple and proteolysis were estimated by reference to the relative mobilities of supplemented with ornithine (2%) or lysine (2%). After incubation standard proteins. The results corresponding to each band were at 37uC for 72 h with shaking, the biogenic amines in the culture expressed as a percentage of total absorbance at 550 nm calculated medium were quantified by high-performance liquid chromatog- with Quantity One Software (Bio-Rad). raphy methods (28). The quantity of each biogenic amine was expressed in parts per million. Lipolytic activity. Lipolytic activity was assessed using the method described by Vignolo et al. (43) with some modifications. Statistical analysis. All statistical analyses were performed One milliliter of a cell suspension of each strain was inoculated using the computer program Statistica 8.0 for Windows (Statsoft into 30 ml of a sterile broth containing 1% (wt/vol) peptone, 0.5% Inc., Tulsa, OK). Significant differences were determined based on (wt/vol) yeast extract, 0.5% (wt/vol) meat extract, and 3% (wt/vol) a one-way analysis of variance. Duncan’s test was used to identify NaCl, pH 7.0, supplemented with 15 g of pork fat. After incubation significant differences among strains. Differences were considered at 37uC for 72 h with shaking, the lipolytic activity was measured significant at P , 0.05. by titration. The fat was extracted according to the method of Folch et al. (11), and the free fatty acids were titrated following the RESULTS AND DISCUSSION procedure of Vignolo et al. (43). The free fatty acid concentration was expressed as percentage of oleic acid (31). Table 2 shows the growth and metabolic characteristics The same medium was also used to determine the ability of of the Bacillus strains studied. The temperature and pH the strains to release free fatty acids from triglycerides. After 72 h values for growth chosen for the assays were those more of incubation, the fat in each culture was extracted as described, frequently found during sausage ripening. Because of the and the free fatty acids were separated from the triglycerides in variability in the NaCl concentrations in sausages, high 1608 CACHALDORA ET AL. J. Food Prot., Vol. 77, No. 9

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 10uC. 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 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/9/1605/1687240/0362-028x_jfp-14-145.pdf by guest on 26 September 2021 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. 1a), actin (Fig. 1b), and myosin (Fig. 1c) 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. 1a, 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 FIGURE 1. SDS-PAGE findings for protein hydrolysis by Bacil- protein profile (Fig. 1a, lane B). Of the 19 strains tested, 6 lus strains. (a) Sarcoplasmic proteins. Lane A, purified sarcoplas- B. subtilis (SA50, SB07, SB14, SB17, SB18, and SB26) and mic proteins (GPI, glucose phosphate isomerase; E, enolase; CK, 4 B. amyloliquefaciens (SA35, SA37, SA39, and SA43) creatine phosphate kinase; A, aldolase; GH, glyceraldehyde hydrolyzed all proteins present in the sarcoplasmic protein phosphate dehydrogenase). Lane B, uninoculated control after extract, resulting in complete absence of all the bands on the 72 h of incubation. Lanes C and D, samples containing different gel (Fig. 1a, lane C) and the generation of new peptides (at isolates after 72 h of incubation: lane C, SA50; lane D, SB01. Lane about 52, 44, 40, 30, 27, and ,20 kDa) in some cases. M, molecular weight standard. (b) Actin. Lane A, purified actin Strain SA06 (B. subtilis) hydrolyzed all the proteins except preparation (D, desmin; Act, actin; Tt, troponin T; Tr, tropomy- the protein of about 37 kDa (glyceraldehyde phosphate osin; MLC, myosin light chain; Ti, troponin I). Lanes B and C, samples containing different isolates after 72 h of incubation: lane dehydrogenase) (data not shown). Strains SB01 and SB13 B, SA06; lane C, SA26. Lane M, molecular weight standard. (c) (B. subtilis) hydrolyzed all proteins present in the extract Myosin. Lane A, purified myosin preparation (MHC, myosin heavy except aldolase (42 kDa) (Fig. 1a, lane D). The remaining chain; a-act, alpha-actinin; D, desmin; Act, actin; Tt, troponin T; strains had lower proteolytic activity against the sarcoplas- Tr, tropomyosin; MLC, myosin light chain; Tc, troponin C). Lanes mic proteins (images not shown). B through D, samples containing different isolates after 72 h of For the actin extract, an intense band appeared at incubation: lane B, SB17; lane C, SA39; lane D, SB15. Lane M, approximately 45 kDa (actin), and other bands appeared for molecular weight standard. many other polypeptides at 66, 55 (desmin), 37 (troponin T), 35 (tropomyosin), 25 (myosin light chain), and 24 (troponin T) kDa (Fig. 1b, lane A). The strains studied had J. Food Prot., Vol. 77, No. 9 METABOLIC STUDY OF BACILLUS STRAINS FROM SAUSAGES 1609

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)a Mean SEM Mean SEM SB07 SB13 Significanceb

C10:0 13.25 3.20 8.56 8.56 26.03 27.95 NS C12:0 24.52 1.95 27.16 0.69 29.18 32.04 NS C14:0 22.92 0.59 25.96 1.59 57.59 70.85 *** C15:0 9.29 1.07 11.08 0.33 13.26 15.26 NS C16:0 69.85 6.42 141.64 25.85 603.34 730.12 *** C16:1 22.94 1.75 21.52 2.80 47.71 53.43 *** C17:0 9.47 0.43 15.87 1.29 38.72 45.52 *** C17:1 11.63 0.30 13.89 0.58 20.56 21.65 *** Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/9/1605/1687240/0362-028x_jfp-14-145.pdf by guest on 26 September 2021 C18:0 44.01 3.70 114.39 14.63 295.50 374.25 *** C18:1 n9 77.84 8.11 205.20 21.75 410.29 450.23 *** C18:2 n6 38.17 4.17 87.38 11.50 167.00 208.59 *** C18:3 n3 19.37 1.30 15.81 1.19 23.35 25.37 NS C20:0 19.60 0.39 21.32 0.34 23.04 26.68 ** C20:1 n9 12.09 0.37 16.47 0.70 20.02 22.28 *** C20:2 n6 11.64 0.98 16.45 0.72 19.25 21.24 * C20:4 n6 11.78 0.26 12.83 0.60 12.52 13.93 NS C22:0 5.17 2.28 9.66 5.64 19.98 21.54 NS C22:2 n6 7.63 1.60 12.32 0.27 15.92 19.78 NS C24:0 12.60 3.05 22.36 0.54 26.03 32.11 NS SFA 236.62 14.00 397.99 38.10 1,131.70 1,376.32 *** UFA 207.14 14.16 401.87 27.54 736.63 836.50 *** MUFA 124.50 8.81 257.09 20.65 498.58 547.59 *** PUFA 82.64 5.83 144.78 13.66 238.05 288.91 *** Total 443.76 26.70 799.86 61.07 1,868.33 2,212.82 *** 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. b 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 SB14, SB15, and SB16. low activity against actin. Only one strain of B. amyloli- been previously reported regarding the proteolytic activity quefaciens (SA26) had an actin band with reduced intensity of Bacillus strains isolated from meat products as deter- (Fig. 1b, lane C). No proteolytic changes were observed for mined by SDS-PAGE techniques nor regarding the the rest of the strains (Fig. 1b, lane B) or in the uninoculated separated protein fractions (sarcoplasmic proteins, actin, control. and myosin). For the myosin extracts, an intense band appeared at Concerning lipolytic activity, the titration method approximately 200 kDa (myosin heavy chain), and other (Table 1) revealed that only two strains of B. subtilis had bands appeared for many other proteins at 102 (alpha- significant lipolytic activity (13.86 and 21.19% of oleic actinin), 55 (desmin), 45 (actin), 37 (troponin T), 35 acid, respectively). These results are not totally in agreement (tropomyosin), 25 (myosin light chain), 20 (troponin C), and with those reported by Baruzzi et al. (3) in Bacillus strains 12 kDa (Fig. 1c, lane A). Of the 19 strains tested, only 2 isolated from Italian sausages and by Ouoba et al. (33) in strains of B. subtilis (SB17 and SB26) did not have Bacillus strains isolated from a fermented African locust proteolytic activity against the myosin (Fig 1c, lane B). bean condiment, which indicates that lipolytic activity Four strains of B. subtilis (SA06, SA50, SB14, and SB18) depends on the strain studied and its source. The strains with and two of B. amyloliquefaciens (SA39 and SA43) totally the highest lipolytic activity also had the highest values for hydrolyzed the myosin heavy chain (Fig. 1c, lane C). The total free fatty acids released, resulting in a significant actions of strains SA28, SB07, and SB16 (B. subtilis) and positive correlation (r ~ 0.92; P , 0.001) between values SA35 (B. amyloliquefaciens) resulted in total hydrolysis of of percentage of oleic acid and total free fatty acid the myosin heavy chain and the generation of new peptides concentrations. (at about 50, 46, and 42 kDa) (data not shown), whereas for Table 3 shows the concentrations of the various fatty the remaining strains the intensity of this protein band was acids freed after 72 h of incubation quantified using gas reduced (Fig. 1c, lane D). These findings cannot be chromatography techniques. Strains were grouped accord- compared with those of other studies because no data have ing to their lipolytic activity: low (,600 mg/100 g of fat), 1610 CACHALDORA ET AL. J. Food Prot., Vol. 77, No. 9 intermediate (700 to 1,000 mg/100 g of fat), and high 3.27 ppm for putrescine and 0.53 to 3.07 ppm for (.1,500 mg/100 g of fat). When comparing low versus cadaverine. No data regarding the amount of biogenic intermediate versus high activity strains, significant differ- amines produced by Bacillus strains of food origin have ences (P , 0.001) were found regarding the sum of been published. Results of the present work indicate that saturated fatty acids, sum of monounsaturated fatty acids, amino acid decarboxylase activity is not particularly high in sum of polyunsaturated fatty acids, and total fatty acids Bacillus strains, especially when compared with other freed. For strains SB07 and SB13 with high lipolytic microbial groups such as lactic acid bacteria or Enterobac- activity, the total free fatty acids were 1,868.33 and teriaceae present in fermented meat products (4, 28). 2,212.82 mg/100 g of fat, respectively, and the saturated In the present study, strains of B. subtilis and B. fatty acids were the main fraction, accounting for more than amyloliquefaciens grew under the salt and pH environmen- 60% of the total free fatty acids. Palmitic acid (C16:0) was tal conditions typically found in sausages during ripening; the main fatty acid (32%), followed by oleic (C18:1 n9), however, only 9 of the 19 strains were able to grow at low stearic (C18:0), and linoleic (C18:2 n6) acids. In the temperatures. All tested strains had proteolytic activity Downloaded from http://meridian.allenpress.com/jfp/article-pdf/77/9/1605/1687240/0362-028x_jfp-14-145.pdf by guest on 26 September 2021 remaining strains, with intermediate or low lipolytic against sarcoplasmic proteins and myosin, and two strains of activity, the percentages for the saturated fatty acids and B. subtilis also had significant lipolytic activity. These unsaturated fatty acids were similar, and the monounsatu- findings indicate that Bacillus strains may play an important rated fatty acids were predominant within the unsaturated role in the ripening of these sausages and in the fatty acids. These strains had similar profiles for free fatty development of their sensory properties. acids; oleic acid was the main fatty acid followed by Among the strains characterized in the present study, B. palmitic, stearic, and linoleic acids. amyloliquefaciens SA35 (isolated from Androlla) and B. These Bacillus strains differed in both their lipolytic subtilis SB07 (isolated from Botillo) grew under the activity (amount of fatty acid freed) and the kind of free environmental conditions tested, hydrolyzed sarcoplasmic fatty acids released. Triglycerides in pork subcutaneous fat proteins and myosin, and had lipolytic activity (very high have a characteristic distribution of fatty acid molecules. activity in SB07 and significant activity in SA35). Both Most of the stearic acid (about 60%) is esterifying the sn1 strains had low levels of decarboxylase activity. In future position of the glycerol, and most of the palmitic acid (60 to work, these two strains will be inoculated into experimental 80 ) is esterifying the sn2 position; most of the oleic and % sausages to study their effects on the physicochemical, linoleic acids (50 to 60%) are in position sn3 and less than microbiological, and sensory characteristics of the final 30% are in position sn1 (2, 7, 22). The free fatty acid product, thus helping to elucidate definitively the role of profiles in these Bacillus strains reflect the specificity of these Bacillus strains in the ripening process of fermented their lipases for the three positions of the triglyceride sausages. molecule. Regarding decarboxylase activity, which was tested ACKNOWLEDGMENTS with agar plate method (Table 2), of the 19 Bacillus strains tested, 73.7% decarboxylated histidine, 94.7% decarboxyl- This work was financially supported by the Xunta de (Regional Government) (project 07TAL021383PR). S. Fonseca acknowl- ated tyrosine, and 73.7% decarboxylated ornithine and edges financial support from the Spanish Ministry of Science and lysine, indicating the potential for these strains to produce Innovation through a predoctoral FPU fellowship (AP2008-03385). The biogenic amines. 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