Letters in 4pplird Microhroloyy 1992. 14. 153-157

Lipolytic activity of Brochothrix thermosphacta on natural trig1 ycerides

R E G I N E T A I O N , M A R T I N E P A P O N , D. B A U C H A R T * , FRANCOISE DUBOISSET* & M A R I E - C H R I S T I N E M O N T E L Station de Recherches sur la Viande, *U.R. Mktabolismes Energktigue er Lipidique, Institut National de la Recherche Agronomique, Centre Clermont- Theiu, 63122 Saint-Gents-Champanelle, France

MS1255: received 13 December I991 and accepted 19 December 1991

T A L O U . R . , P A P O \ . M.. B A I J C H A R T . D., DUBOISSET, F. & M O N T E L , M.-c 1992. Lipolytic activity of Brochothriv thermosphacta on natural triglycerides. Letters in Applied 1l4icrohioloyy 14. 153-157.

Four natural gllcerides were used to study the lipolytic characteristics of Brocho- thrix thermosphuc t u lipase. Hydrolysis of triglycerides from linseed oil and pork fat adipose tissue with a high percentage of unsaturated fatty acids was two to three times greater than in saturated triglycerides from lamb or beef fat adipose tissues. Hydrolysis was not specific as the percentage of the released fatty acids was similar to that of the starting triglycerides.

Brochothrix thermosphacta is an important spoilage bacterium commonly found in meat and meat products. It has been isolated from beef carcasses during dressing, chilling and boning (Newton p r a / . 1978), and from beef refrigerated under oxygen-permeable conditions and in modified atmospheres (Patterson & Gibbs 1977; Dainty er a / . 1979). It often domi- nates the microflora of vacuum-packed lamb and may constitute 60-70% of the flora (Gill & Newton 1977; Shaw et a / . 1980). In fresh and cured pork and in sausage Broc. thermosphacta accounted for a large part of the flora (Gardner 1981).

Brochothrix rherrnosphactci grows rapidly in adipose tissue (Grau 1983: Talon & Monte1 1986) and may reach lo9 cells,'g. Off flavours were detected at 10' cells'g (Egan & Grau 1981). This spoilage can be due to the pro- duction of volatile fatty acids (Dainty 1981). The precursors of acetic, isobutyric and isovaleric acids are glucose and amino acids but for butyric and propionic acids the origin is still unknown (Dainty & Hibbard 1983). Other vola- tile compounds ( 2 methyl propanol, 3 methyl butanol and 3 methyl butanal) are associated

with the spoilage of vacuum-packaged luncheon meat by Broc. thermosphacta but the metabolic origins of these compounds are not known (Stanley et al. 1981).

A previous study has shown that Broc. t h o - mosphacra lipase preferentially hydrolyses tribu- tyrin and its activity decreases as the length of the carbon chain fatty acids increases (Papon & Talon 1989). So far, natural triglycerides whose degree of saturation and position of fatty acids are different have not been studied. 'The purpose of the present study was to investigate the lipo- lytic action of Broc. thermosphactu against natural fats.

Materials and Methods

L I P A S F P R F P A R A r Io \

Brochothrix thermosphac ta (ATCC 1 1509) WAS

grown In Bacto APT broth (Difco) at 24 C Cells in exponential growth were harvested by centrifugation at 6000 g for 10 mln at 4°C The cells (0 25 g wet weight/ml) were resuspended in

20 mmol/l Tris-HC1, 10 mmol/l CaCI, buffer pH

Regine Talon et al. Table 1. Fattv acid comDosition of trialwerides used as substrates

~~ ~ ~ ~

Pork fat Beef fat Lamb fat Linseed oil Fatty acid (Yo) (%) (%) ("/.I

0.2 c12:o -

C14:O 0.9 2.3 1.6 0.1 C15:O 0.1 0.7 0.6 C16:O 18.5 23.9 20.5 4.5 C 16 : l(n-7) 1.4 1.6 1.2 C17:l(n-8) 0.3 0.6 0.6 C18:O 12.2 25.5 30.6 4.6 C18:l(n-9) 47.1 40.3 40.9 15.2 C18 :2(n-6) 14.7 3.1 1.3 16.1 C18:3(n-3) 2.8 1.1 1.8 57.7 c20:o 0.2 0.2 0.3 0.2 C20:l(n-11) 1.5 0.4 0.6 0.3 Saturated FA 32.2 53.0 53.6 10.6 Monounsaturated FA 50.3 42.9 43.3 15.6 Polyunsaturated FA 17.5 4.2 3.1 73.8

FA, Fatty acid.

- 0.1

-

- -

7.0 (TCB) and ultrasonically disrupted in an ice T R I G L Y C E R I D E P U R I F I C A T I O N

bath under nitrogen with a Vibra-Eel1 (Bioblock) as described by Papon & Talon (1989). Cell-wall material and unbroken cells were separated by centrifugation at 2 5 0 0 0 g for 30 min at 4°C. Proteins of the supernatant fluid were precipi- tated with ammonium sulphate (60% saturation) and centrifuged at 75000 g for 30 min at 4°C. The resulting precipitate was dis- solved in TCB (7.0 mg proteins/ml) and dialysed against the same buffer for 15 h at 4°C.

Natural triglycerides from fat tissues of pork, beef and lamb chops and from linseed oil were used. The lipids were extracted according to the method of Folch et a!. (1957). The triglycerides were purified by liquid-chromatography on a Florisil column (lipid-free Florisil, Toward and Matignon, 60-100 mesh) with hexane with 4% diethyl ether as an eluting solvent (Christie 1973).

Table 2. Release of free fatty acids from triglycerides by Brochothrix therrnosphacta lipase after 24 h incu- bation at 37°C

Pork fat Beef fat Lamb fat Linseed oil

Fatty acid PdP Y" PP/g Yo PCpk % PdP '%

- - C12:O 1.1 0.1 1.2 0.1 0.2 0.3 C14:O 15.5 1 .o 13.5 1.7 23.9 3.6 C15:O 7.1 0.5 4.5 0.6 6.1 1 .0 2.9 0.1 C16:O 209.8 13.8 161.2 20.5 139.4 21.1 122.0 5.8 C16:l(n-7) 26.1 1.7 12.3 1.6 15.8 2.3 20.0 0.9 C17:1(n-8) C18:O 200.0 13.1 237.2 30.3 209.1 31.7 44.9 2.1

756.0 49.6 343.6 43.9 209.8 31.8 350.1 16.7 C 18 : 1 (n-9) 0.3 ~ - 282.9 13.4 C18 :2(n-6) 264.6 17.3 2.0

C18:3(n-3) 45.2 3.0 6.7 0.9 54.8 8.3 1273.3 60.7 c20:o C20: l(n-11) - - Total* 1525.4 100.0 781.8 100.0 659.7 100.0 2096.1 100.0

Saturated FA 433.5 28.5 417.6 53.4 379.3 57.7 169.8 8.0 Monounsaturated FA 782.1 51.3 355.5 45.5 225.6 34.1 370.1 17.6 Polyunsaturated FA 309.8 20.3 8.7 1.1 54.8 8.3 1556.2 74.3

- -

~ - - - -

~ ~ - - - - - - ~ ~ - - - ~

k 50.0 & 50.0 k 40.0 * 100.0

* Average of four values. FA, Fatty acid.

Lipolytic activity of Broc. thermosphacta 155

Purity of the triglycerides was checked by thin-layer chromatography on Kieselgel G (Merck, Darmstadt, Germany). The plates were developed with hexane-diethyl ether-formic acid (120/60/1.5, v 'vjv). Lipid classes, stained with iodine vapours, were identified with reference to standard lipid mixtures.

After saponifying all lipids in 10% KOH, the fatty acids were methylated with 3% HCI meth- anol. Fatty acid methyl esters of triglycerides were analysed by gas liquid-chromatography with a glass capillary column coated with free fatty acid phase (FFAP) and the following chro- matographic conditions: oven temperature at lYS"C, injector and detector temperatures at 250"C, gas carrier: helium (Bauchart & Aurousseau 1981). Fatty acids were identified by comparing their equivalent chain length (ECL) with standard commercial fatty acids.

R E A C T I O N M l Y l L R E

Gum arabic lo", , v, v and 20% of pure tri- glyceride (w/v) in water were emulsified by soni- cation for 3 min at room temperature.

A reaction mixture was produced by mixing: 2.2 ml of fat emulsion, 1.1 ml of I mol:l Tris- HCI buffer pH 7.0 with 0.08 molil CaCI, , 1. I ml of 1.0 mol 1 NaCI, 2.2 ml of enzyme prep- aration, and 4.4 ml of distilled water. Controls containing enzyme without substrate and sub- strate without enzyme were similarly treated.

The reaction mixtures were incubated by shaking for 24 h at 31 C.

E X T R A C T I O N A X I I ) A S A L Y S I S O F F R E k

F A T T Y AC'IIIS (FFA)

After hydrolysis. the FFA were extracted according to the method of Needs et al. (1983). Lipid was extracted in ether and FFA recovered by shaking the extract with anion exchange resin Amberlyst A26. The FFA were methylated directly and released from the resin with ether and saturated NaC1. The individual acids were quantified with internal standards by gas chro- matography as described above.

Results were expressed as pg of the released fatty acid/& of lipids.

Results

The fatty acid composition of difrerent tri- glycerides used as lipid substrates for Broc. ther-

mosphacta is summarized in Table 1. Unsaturated fatty acids represented 66% in pork triglyceride and 89% in linseed oil. Oleic and linolenic acids respectively, were predomi- nant in pork fat and linseed oil. For beef and lamb triglycerides, the proportion of saturated and unsaturated fatty acids was similar with oleic acid predominating. There were also large amounts of stearic and palmitic acids.

After incubation at 37-C for 24 h, the tri- glycerides were hydrolysed by Broc. ther- mosphacta lipase. The two substrates with the higher percentage of unsaturated fatty acids (linseed oil and pork fat) were more rapidly hydrolysed (2.1 mg FFAig and 1.5 mg FFA/g) than lamb and beef fats (0.7-0.8 mg FFA/g) (Table2).

Brochothrix therrnosphacta lipase prefer- entially released linolenic acid from linseed oil, oleic acid from pork and oleic and stearic acids from beef and lamb (Table 2). During the lipolysis of the three animal fats, release of pal- mitic acid was also substantial. Hence, the per- centage of the resulting fatty acids was very similar to that of the initial substrates (Tables 1 and 2).

Discussion

The rates of fatty acid production by Broc. ther- rnosphactu lipase are comparable with those of lipolysis of subcutaneous adipose pork tissues by Lirctobacillus curmius and Pediococcus pen- tosaceus, which were 10 mg acid/& of lipid within 8 d at 37 C (Nieto et al. 1989). Brocho- thrix therrnosphactu preferentially hydrolysed pork fat. whereas the activities of sausage starter cultures were higher in lamb and beef kidney fat than in pork kidney fat (Nielsen & Kemner 1989). However, the lipolytic effects of these bac- teria depended on the origin of the pork fat. The action was higher in back fat than in fat from the kidney region.

Brochothris thermosphucra lipase released the fatty acids randomly. These results are in accordance with those of Debevere et al. (1976) in respect of the lipolytic activity of Micrococcus sp. in pork fat. In addition, the pattern of fatty acids released by Broc. therrnosphacta from pork fat proved to be similar to those of Micrococcus spp., Staphylococcus .ujlo.sus and Lactobacillus plantmum on this substrate (Cantoni et a/. 1967; Nieto et ul. 1989). However, Nieto et al. (1989)

156 Regine Talon et al.

noted that palmitic acid was the major com- ponent (ca 40%) released from beef and lamb fats by all the strains, whereas this acid rep- resented only 20% of those released by Broc. therrnosphacta. Positional and fatty acids spe- cificities are described for some microbial lipases (Alford et al. 1971; Jensen et a/ . 1983). Walhroos & Niinivaara (1969) have mentioned a greater increase in unsaturated acids compared with saturated acids during lipolysis in the sausage ripening process. Cerise et a / . (1972) and Demeyer et a / . (1974) stated that the increase in oleic acid observed during sausage ripening was due to specific lipolytic attack on the 1 and 3 positions of the triglyceride, which are prefer- entially occupied by this acid in animal fats. Nestorov et a/. (1983) have drawn the same con- clusion about a yeast starter culture preferring unsaturated fatty acids.

In conclusion, the lipase of Broc. ther- rnosphacta hydrolyses natural triglycerides poorly compared with tributyrin. The activity is reduced at 8°C and at pH 5.8 (Papon & Talon 1989). Thus in pH range of meat and at refriger- ation temperature, activity would be limited. In fermented sausage Broc. therrnosphacta has fre- quently been isolated but only in small numbers us lactic acid bacteria and staphylococci. Its role in flavour development would therefore be slight.

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