EFFECTS OF STAPHYLOCOCCUS CONDIMENTI AND
MICROCOCCUS CASEOLYTICUS ON THE VOLATILE
COMPOUNDS OF CANTONESE SAUSAGE
YANTAO WU 1, CHUN CUI 1, WEIZHENG SUN 1, BAO YANG 2and
MOUMING ZHAO 1,3
1College of Light Industry and Food Sciences
South China University of Technology
Guangzhou 510640, China
South China Botanical Garden
Chinese Academy of Sciences
Guangzhou Leyiju 510650, China
Accepted for Publication December 29, 20072
ABSTRACT
Two strains, Staphylococcus condimenti and Micrococcus caseolyticus, were screened and isolated from Cantonese sausage in this study. The effects of these two strains on the flavorof Cantonese sausage were also investigated. Three Cantonese sausage samples were prepared:control, sausage inoculated with S. condimenti (SC)and sausage inoculated with M. caseolyticus (MC).The volatile compounds were extracted by a headspace solid-phase microextraction method, quantifiedand identifiedby gas chromatography–massspectrometry. A total of 22, 22and 25volatile com-pounds were identifiedfrom the three samples, respectively. Larger amounts of volatile compounds (alcohols,aldehydes, ketones and esters) were detected in SC and MC, particularly for aldehydes and ethyl esters, whereas the amounts of hydrocarbons and heterocyclic compounds decreased. The results showed that both strains could accelerate the degradation and oxidation of lipids and proteins, and improve the flavorcharacteristics of Cantonese sausage.
PRACTICAL APPLICATIONS
Cantonese sausage is a traditional Chinese meat product that is well accepted by consumers over the world. In order to improve the flavor3Corresponding author. TEL:+86-20-87113914; FAX:+86-20-87113914; EMAIL:[email protected]
Journal of Food Process Engineering 32(2009)844–854.All Rights Reserved.
Copyright the Authors
Journal Compilation 2008Wiley Periodicals, Inc.
DOI:10.1111/j.1745-4530.2008.00249.x844
EFFECTS OF S. CONDIMENTI AND M. CASEOLYTICUS ON CANTONESE SAUSAGE 845characteristics of this sausage, two strains, Staphylococcus condimenti and Micrococcus caseolyticus , were used as starter cultures for Cantonese sausage preparation in this work. The volatile compounds were identifiedby gas chromatography–massspectrometry. The results showed that these two strains could be good flavorenhancers of Cantonese sausage. Therefore, application of S. condimenti or M. caseolyticus on the Cantonese sausage will be of great significancein improving the quality of this traditional food.
INTRODUCTION
Cantonese sausage has gained much popularity and acceptance all over the world, because of its unique flavorand taste. Cantonese sausage is a semi-dry sausage whose flavoris the result of a complex combination of hydrocarbons, aldehydes, ketones, alcohols and esters (Duand Ahn 2001). Many of these compounds are formed by enzymatic reactions (glycolysis,proteolysis, oxidative deamination, transamination and decarboxylation) or chemical processes (lipidautoxidation, Strecker degradation, Maillard reac-tion) taking place during the processing, ripening and storage of sausages (Montelet al . 1998; Ordóñezet al . 1999; Stahnke 1999).
Microbial growth in sausages has been considered an important factor in the formation of volatile compounds. Recently, the effect of starter cultures on the production of volatile compounds has been extensively studied (Berdaguet al . 1993; Johansson et al . 1994; Stahnke 1994; Montel et al . 1996). Many starter cultures of bacteria, yeast and fungi are commonly used as flavorenhancers in industrial fermentation. Staphylococci are used as a starter culture in sausage production in combination with lactic acid bacteria, because they are considered two important species in the production of flavorcom-pounds (Stahnke1999). Montel et al . (1996)have found that both Staphylo-coccus carnosus and Staphylococcus xylosus produce the highest amounts of branched aldehydes, methyl ketones and ethyl esters, producing the highest scores for cured odor. Stahnke (1999)has also observed that S. carnosus and S. xylosus produce a great number of volatile compounds in concentrations that are of sensory importance. To date, there are still limited publications on Staphylococcus ondimenti and Micrococcus caseolyticus , especially in meat products.
In this study, S. condimenti and M. caseolyticus were screened and iso-lated for the firsttime from Cantonese sausage. Their effects on the flavorcharacteristics of Cantonese sausage were also investigated by solid-phase microextraction (SPME)and gas chromatography–massspectrometry (GC–MS) techniques.
846Y . WU ET AL.
MATERIALS AND METHODS
Materials
Cantonese sausage was prepared according to the following formulation:lean pork (70g) and back fat (30g), salt (3.5g), sugar (6g), wine (4g), sodium nitrite (0.02g), water (20g). These raw materials were mixed and stuffed into casings with a diameter of 37mm and oven-dried for 6h at 55C, followed by a reduction of temperature (from55to 50C), then oven-dried for another 66h at 50C. The control Cantonese sausage was obtained. Samples were inoculated with S. condimenti (SC)and M. caseolyticus (MC)at 106cfu/g,respectively. After drying, the products were vacuum-packaged and stored at –20Cuntil analysis.
Isolation and Identificationof Bacteria
Staphylococci were isolated from Cantonese sausages. The strains were selected by standard microbiological methods using appropriate dilutions in buffered peptone solution (Simonováet al . 2006). Dilutions were plated onto mannitol salt agar plates and incubated at 37C for 48h. After incubation, the colony forming units were counted. Among gram-and catalase-positive strains, 64belonged to the staphylococci group (64strains). According to the sensory evaluation, four strains producing aroma were selected for further identificationand testing. They were further characterized by the following complementary biochemical tests:tolerance to NaCl and nitrite, acid produc-tion, adhesion assay, and growth ability, lipolytic activity and proteolysis activity by the methods of Papamanoli et al . (2002),Rantsiou et al . (2005)and Simonováet al . (2006).Two strains were confirmedsuitable to be starter cultures in Cantonese sausages. They were further identifiedby the means of polymerase chain reaction-denaturing gradient gel electrophoresis described by Rantsiou et al . (2005).
Analysis of Volatile Compounds
SPME Fibers. V olatile compounds were extracted by SPME method. An SPME device (Supelco,Bellefone, PA) was used in this study, with 50m m of divinylbenzene/carboxen/polydimethylsiloxanefiber.Before the analysis, the fiberwas preconditioned in the injection port of GC as indicated by the manufacturer.
Procedure
For each experiment, 5g of Cantonese sausage was minced and weighed into a 50-mL headspace vial and sealed with a Teflonsilicone septum. The vial
EFFECTS OF S. CONDIMENTI AND M. CASEOLYTICUS ON CANTONESE SAUSAGE 847was left at 30C in a thermal block for 1h to equilibrate the headspace. Then, an SPME fiberwas exposed to the headspace while maintaining the sample for 60min. The compounds absorbed by the fiberwere identifiedand quantifiedby GC–MS.
Identificationand Quantificationof Volatile Compounds
The flavorcompounds adsorbed by the fiberwere desorbed in the injec-tion port of GC–MS(FinniganMAT 8230GC-MS, Palo Alto, CA) for 3min at 230C with a splitless injection mode. The compounds were separated in a DB-1capillary column (30m ¥0.25mm internal diameter, 1m m of filmthickness). Helium was used as carrier gas with a linear velocity of 1mL/min.The GC oven temperature program was started when the fiberwas inserted and held at 50C for 3min, ramped to 120C at 5C/min,holding for 2min, and then to 220C at 10C/min,holding for 10min. The transfer line to the mass spec-trometer was maintained at 230C. The mass spectra were obtained using a mass selective detector by electronic impact at 70eV . The detector voltage was 350V , and the scan range of m/zwas 35–395amu.
Identificationof flavorcompounds was carried out according to the reten-tion time and by comparing mass spectrum data (RT/MS)of the samples with the the National Institute of Standards and Technology literature database. V olatile compounds were quantifiedby calculating the peak area.
RESULTS AND DISCUSSION
Identificationand Characterization of Strains
The DNA sequences were analyzed. The comparison for DNA similarity was performed with the GenBank, European Molecular Biology Laboratory, DNA Databank of Japan and Protein Data Bank database (Blaiottaet al . 2003). Through this analysis, two strains were identifiedas S. condimenti and M. caseolyticus , respectively.
Analysis of Volatile Compounds
The three samples had 22, 22and 25volatile compounds, respectively. Compounds identifiedin the samples are listed in Tables 1–3.Their origins are also listed. Four hydrocarbons, two alcohols, fivealdehydes, one ketones, eight esters and two heterocyclic compounds were identifiedin control; two alcohols, six aldehydes, one ketones, 10esters and three heterocyclic com-pounds were found in SC; one hydrocarbon, four alcohols, seven aldehydes, three ketones, eight esters and three heterocyclic compounds were observed in
848Y . WU ET AL.
TABLE 1.
VOLATILE COMPOUNDS IDENTIFIED IN CANTONESE CONTROL SAUSAGES AND
THEIR ORIGINS
Number IdentificationCompound
Ethanol
1-octen-3-ol
Pentanal
Hexanal
Heptanal
2-octenal
Nonanal
2,3-octanedione
Propanoic acid, ethyl ester
1-butanol, 3-methyl-, formate
Butanoic acid, ethyl ester
Propanoic acid, 2-hydroxy, ethyl ester
Butanoic acid, 3-methyl-, ethyl ester
Pentanoic acid, ethyl ester
Hexanoic acid, ethyl ester
Octanoic acid, ethyl ester Area (%)47.990.900.745.150.350.231.281.660.492.831.451.750.460.234.361.05
5.52
5.49
0.14
0.10
10.70
0.21Origin Wine Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Microbial Microbial Microbial Microbial Microbial Microbial Microbial Microbial Unknown Lipid Unknown Unknown Unknown Contaminant Alcohols (48.89%)1RT/MS2RT/MSAldehydes (7.75%)3RT/MS4RT/MS5RT/MS6RT/MS7RT/MSKetones (1.66%)8RT/MSEsters (12.62%)9RT/MS10RT/MS11RT/MS12RT/MS13RT/MS14RT/MS15RT/MS16RT/MSAlkanes and alkenes (11.25%)17RT/MSMethylene chloride 18RT/MSHexane 19RT/MSn-ethyl-1,3-dithioisoindoline 20RT/MSn-ethyl-1,3-dithioisoindoline Heterocyclic compounds (10.91%)21RT/MSBenzene 22RT/MSToluene
RT/MS,retention time and mass spectrum data.
MC. Cantonese sausages inoculated with starter cultures had higher alcohols, aldehydes, ketones and esters as well as lower hydrocarbon and cyclic com-pounds than the control.
SC and MC showed higher levels of alcohols than the control, especially for 2-pentanol and 2-(2-butoxyethoxy)-ethanol,including ethanol and 1-octen-3-ol in MC. This result could be attributed to the starter culture-related fermentation and lipid oxidation. The results showed that ethanol was the most abundant desorbed volatile compound. In the control, 47.99%of ethanols were determined, while in SC and MC, 53.00and 46.16%of ethanols were
EFFECTS OF S. CONDIMENTI AND M. CASEOLYTICUS ON CANTONESE SAUSAGE 849
TABLE 2.
VOLATILE COMPOUNDS IDENTIFIED IN CANTONESE STAPHYLOCOCCUS CONDIMENTI
SAUSAGES AND THEIR ORIGINS
Number IdentificationCompound
Ethanol
1-octen-3-ol
Pentanal
Hexanal
Heptanal
2-heptenal
2-octenal
Nonanal
2,3-octanedione
Acetic acid, ethyl ester
Propanoic acid, ethyl ester
1-butanol, 3-methyl-, formate
butanoic acid, ethyl ester
Ethyl 2-hydroxy-propanoate
Butanoic acid, 2-methyl-, ethyl ester
Butanoic acid, 3-methyl-, ethyl ester
Pentanoic acid, ethyl ester
Hexanoic acid, ethyl ester
Octanoic acid, ethyl ester Area (%)53.001.440.888.640.430.590.471.442.733.270.503.232.172.490.230.630.324.791.22
4.61
0.22
0.10Origin Wine Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Fermentation Microbial Microbial Microbial Fermentation Microbial Microbial Microbial Microbial Microbial Unknown Contaminant Unknown Alcohols (54.44%)1RT/MS2RT/MSAldehydes (12.45%)3RT/MS4RT/MS5RT/MS6RT/MS7RT/MS8RT/MSKetones (2.73%)9838Esters (18.85%)10RT/MS11RT/MS12RT/MS13RT/MS14RT/MS15RT/MS16RT/MS17RT/MS18RT/MS19RT/MSHeterocyclic compounds (4.81%)20RT/MSBenzene 21RT/MSToluene 22RT/MS1,2-dihydro-1,4-diphenylphtyalazine
RT/MS,retention time and mass spectrum data.
identified,respectively. A similar result was reported by Du and Ahn (2001).These authors also suggested that wine is responsible for the development of flavorin Cantonese sausages. A large amount of wine (approximately4%in comparison with the weight of meat) is usually added at the beginning of the sausage-making process. This leads to a higher proportion of ethanol and other volatile compounds in the sausage.
Many researchers have studied the role of starter culture in the production of aldehydes and have suggested that species of the genus Staphylococcus are more important than Lactobacillus and Pediococcus in relation to the produc-tion of volatile compounds (Berdaguet al . 1993). From the data in Tables 1–3,aldehydes account for 7.75, 12.45and 13.1%of the total volatile compounds
850Y . WU ET AL.
TABLE 3.
VOLATILE COMPOUNDS IDENTIFIED IN CANTONESE MICROCOCCUS CASEOLYTICUS
SAUSAGES AND THEIR ORIGINS
Number IdentificationCompound
Ethanol
2-pentanol
1-octen-3-ol
2-(2-butoxyethoxy)-ethanol
Pentanal
Hexanal
Heptanal
2-octenal
Nonanal
2-nonenal
6-(hydroxyl-phenyl-methyl)-2,2-dimethyl-
Cyclohexanone
1-octen-3-one
2,3-octanedione
Acetic acid, ethyl ester
Butanoic acid, ethyl ester
Ethyl 2-hydroxy-propanoate
Butanoic acid, 2-methyl-, ethyl ester
Butanoic acid, 3-methyl-, ethyl ester
Pentanoic acid, ethyl ester
Hexanoic acid, ethyl ester
Octanoic acid, ethyl ester Area (%)46.163.471.563.631.088.530.420.511.820.150.780.142.732.752.232.570.210.540.336.602.20
0.15
3.33
0.18
0.18Origin Wine Fermentation Lipid oxidation Fermentation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Alcohols (54.82%)1RT/MS2RT/MS3RT/MS4RT/MSAldehydes (13.1%)5RT/MS6RT/MS7RT/MS8RT/MS9RT/MS10RT/MSKetones (3.65%)11RT/MS1213RT/MSRT/MSLipid oxidation Fermentation Microbial Fermentation Microbial Microbial Microbial Microbial Microbial Fermentation Unknown Contaminant Unknown Esters (17.43%)14RT/MS15RT/MS16RT/MS17RT/MS18RT/MS19RT/MS20RT/MS21RT/MSAlkanes and alkenes (0.15%)22RT/MSL-limonene Heterocyclic compounds (3.69%)23RT/MSBenzene 24RT/MSToluene 25RT/MS2-pentyl-furan
RT/MS,retention time and mass spectrum data.
in the control, SC and MC sausages, respectively. Aldehydes in Cantonese sausages were linear aliphatic from C 4up to C 10. Among these, hexanal and nonanal were the main aldehydes found in Cantonese sausage. This result is similar to that reported by Meynier et al . (1999).Hexanal has been suggested as the oxidation product of linoleic acid and the detection of hexanal indicates the degree of lipid oxidation in the sausage (Duand Ahn 2001). SC and MC
EFFECTS OF S. CONDIMENTI AND M. CASEOLYTICUS ON CANTONESE SAUSAGE 851sausages showed higher levels of aldehydes than the control, especially for hexanal and nonanal. These aldehydes are produced during the oxidative degradation of unsaturated fatty acids (Mottram1998). After inoculating the two strains, the oxidative degradation of unsaturated fatty acids increased because of the lipolytic activity of S. condimenti and M. caseolyticus . Besides aldehydes, there are many other volatile compounds arising through lipid oxidation, such as hydrocarbons, alcohols and ketones. They were present in small proportions compared with aldehydes. SC and MC sausages had larger amounts of hydrocarbons, alcohols and ketones.
As a whole, volatile compounds generated by lipid oxidation accounted for 10.31%of the total volatile compounds of control sausages, which was lower than those for the samples inoculated with starter cultures (16.62%in SC and 23.85%in MC). These results are consistent with the biochemical char-acteristics of S. condimenti and M. caseolyticus , which possess lipolytic activ-ity and the function of oxidative deamination and transamination (Klooset al . 1998; Probst et al . 1998).
Esters are another group of volatile compounds that showed differences between the control and the sausages inoculated with S. condimenti and M. caseolyticus . In control, SC and MC sausages, 8, 10and 8esters were identified,which accounted for 12.62, 18.85and 17.43%of total volatile compounds, respectively (Tables1–3).The results indicate that S. condimenti and M. caseolyticus could increase the amount of ethyl esters, most of which resulted from the microbial esterificationof acids (derivedfrom sugar fermen-tation, lipolytic activity or degradation and oxidation of amino acid) with alcohols. Our result is in agreement with those previously obtained in dry sausages by Stahnke (1994,1995). Montel et al . (1996)have also found that S. xylosus and S. carnosus are the main producers of ethyl esters, giving rise to sausage models with a better flavor.
In the present study, sausages inoculated with S. condimenti and M. caseolyticus showed higher levels of 3-methyl ethyl butanoate, 2-methyl ethyl butanoate and ethyl acetate than control sausages. This fact might be attributed to the inoculation of Staphylococcus species, which can transform branched amino acids (valine,isoleucine and leucine) during sausage ripening into 3-methy butanal and 2-methyl butanal through a nonenzymatic Strecker reaction (Barbieriet al . 1992; Berdagu et al . 1993) or by microorganism metabolism (Meynieret al . 1999). Then the corresponding alcohols were formed by the reduction of the aldehydes. Or the aldehydes are oxidized to the corresponding acids and esterifiedwith ethanol by Staphylococcus , producing 3-methyl ethyl butanoate and 2-methyl ethyl butanoate (Stahnke1994; Meynier et al . 1999; Hérranzet al . 2005).
Greater amounts of ethyl esters in SC and MC sausages can be consi-dered favorable because they are essential to the overall flavorof fermented
852Y . WU ET AL.
sausages, providing fruity notes or masking rancid odor (Hérranzet al . 2005). Compared with the volatile compounds derived from the lipid degradation and oxidation, esters have a lower proportion in Cantonese sausages, but they play a more important role in the flavorof Cantonese sausages because of their very low threshold value. From the results shown, we inferred that esters are important for the flavorof Cantonese sausages, and 2-hydroxy ethyl pro-panoate is responsible for the unique flavorof Cantonese sausages.
In the present study, benzene and methyl benzene were found in the three samples. Methylene chloride and n-ethyl-1,3-dithioisoindoline were found in control sausages. These volatile compounds did not directly originate from the meat products. Similar results were reported by Du and Ahn (2001).These authors detected benzene, methyl benzene and chloroform in Cantonese sausages, and they pointed out that these volatile compounds are from the packaging materials. Further work will be carried out in order to interpret this phenomenon.
In addition, L-limonene was found in MC. It is probably formed through the extensive degradation of lipids during the ripening of Cantonese sausages after inoculation with M. caseolyticus . 2-pentyl-furan was also found in MC because of the autoxidation caused by M. caseolyticus .
CONCLUSION
From the results of this work, the use of S. condimenti and M. caseolyti-cus is beneficialto the degradation and oxidation of lipid and amino acids. S. condimenti and M. caseolyticus could improve the overall flavorof Can-tonese sausages because of an increase in the generation of volatile com-pounds. The study also indicates that S. condimenti and M. caseolyticus have good potential to be starter cultures in sausages. Unlike in previous reports, branched aldehydes were not detected in the three samples used in the present study. Branched aldehydes may have been converted into their corresponding esters by transamination, oxidative deamination and microbial esterification.This study indicates that the majority of volatile compounds from Cantonese sausages are alcohol. Although volatile compounds generated from fatty acids, amino acids, microorganism and fermentation only account for a small pro-portion, they play an important role in the flavorof Cantonese sausages.
ACKNOWLEDGMENTS
This work was financiallysupported by the Science and Technology Foundation of Guangdong Province (GrantNo. 2005A20303002) and the
EFFECTS OF S. CONDIMENTI AND M. CASEOLYTICUS ON CANTONESE SAUSAGE 853University-Enterprise Partnership Foundation of Guangdong Province (GrantNo. 2006D90202001). The authors want to acknowledge Yufen Chen and Caizhen Yang for their technical help.
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EFFECTS OF STAPHYLOCOCCUS CONDIMENTI AND
MICROCOCCUS CASEOLYTICUS ON THE VOLATILE
COMPOUNDS OF CANTONESE SAUSAGE
YANTAO WU 1, CHUN CUI 1, WEIZHENG SUN 1, BAO YANG 2and
MOUMING ZHAO 1,3
1College of Light Industry and Food Sciences
South China University of Technology
Guangzhou 510640, China
South China Botanical Garden
Chinese Academy of Sciences
Guangzhou Leyiju 510650, China
Accepted for Publication December 29, 20072
ABSTRACT
Two strains, Staphylococcus condimenti and Micrococcus caseolyticus, were screened and isolated from Cantonese sausage in this study. The effects of these two strains on the flavorof Cantonese sausage were also investigated. Three Cantonese sausage samples were prepared:control, sausage inoculated with S. condimenti (SC)and sausage inoculated with M. caseolyticus (MC).The volatile compounds were extracted by a headspace solid-phase microextraction method, quantifiedand identifiedby gas chromatography–massspectrometry. A total of 22, 22and 25volatile com-pounds were identifiedfrom the three samples, respectively. Larger amounts of volatile compounds (alcohols,aldehydes, ketones and esters) were detected in SC and MC, particularly for aldehydes and ethyl esters, whereas the amounts of hydrocarbons and heterocyclic compounds decreased. The results showed that both strains could accelerate the degradation and oxidation of lipids and proteins, and improve the flavorcharacteristics of Cantonese sausage.
PRACTICAL APPLICATIONS
Cantonese sausage is a traditional Chinese meat product that is well accepted by consumers over the world. In order to improve the flavor3Corresponding author. TEL:+86-20-87113914; FAX:+86-20-87113914; EMAIL:[email protected]
Journal of Food Process Engineering 32(2009)844–854.All Rights Reserved.
Copyright the Authors
Journal Compilation 2008Wiley Periodicals, Inc.
DOI:10.1111/j.1745-4530.2008.00249.x844
EFFECTS OF S. CONDIMENTI AND M. CASEOLYTICUS ON CANTONESE SAUSAGE 845characteristics of this sausage, two strains, Staphylococcus condimenti and Micrococcus caseolyticus , were used as starter cultures for Cantonese sausage preparation in this work. The volatile compounds were identifiedby gas chromatography–massspectrometry. The results showed that these two strains could be good flavorenhancers of Cantonese sausage. Therefore, application of S. condimenti or M. caseolyticus on the Cantonese sausage will be of great significancein improving the quality of this traditional food.
INTRODUCTION
Cantonese sausage has gained much popularity and acceptance all over the world, because of its unique flavorand taste. Cantonese sausage is a semi-dry sausage whose flavoris the result of a complex combination of hydrocarbons, aldehydes, ketones, alcohols and esters (Duand Ahn 2001). Many of these compounds are formed by enzymatic reactions (glycolysis,proteolysis, oxidative deamination, transamination and decarboxylation) or chemical processes (lipidautoxidation, Strecker degradation, Maillard reac-tion) taking place during the processing, ripening and storage of sausages (Montelet al . 1998; Ordóñezet al . 1999; Stahnke 1999).
Microbial growth in sausages has been considered an important factor in the formation of volatile compounds. Recently, the effect of starter cultures on the production of volatile compounds has been extensively studied (Berdaguet al . 1993; Johansson et al . 1994; Stahnke 1994; Montel et al . 1996). Many starter cultures of bacteria, yeast and fungi are commonly used as flavorenhancers in industrial fermentation. Staphylococci are used as a starter culture in sausage production in combination with lactic acid bacteria, because they are considered two important species in the production of flavorcom-pounds (Stahnke1999). Montel et al . (1996)have found that both Staphylo-coccus carnosus and Staphylococcus xylosus produce the highest amounts of branched aldehydes, methyl ketones and ethyl esters, producing the highest scores for cured odor. Stahnke (1999)has also observed that S. carnosus and S. xylosus produce a great number of volatile compounds in concentrations that are of sensory importance. To date, there are still limited publications on Staphylococcus ondimenti and Micrococcus caseolyticus , especially in meat products.
In this study, S. condimenti and M. caseolyticus were screened and iso-lated for the firsttime from Cantonese sausage. Their effects on the flavorcharacteristics of Cantonese sausage were also investigated by solid-phase microextraction (SPME)and gas chromatography–massspectrometry (GC–MS) techniques.
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MATERIALS AND METHODS
Materials
Cantonese sausage was prepared according to the following formulation:lean pork (70g) and back fat (30g), salt (3.5g), sugar (6g), wine (4g), sodium nitrite (0.02g), water (20g). These raw materials were mixed and stuffed into casings with a diameter of 37mm and oven-dried for 6h at 55C, followed by a reduction of temperature (from55to 50C), then oven-dried for another 66h at 50C. The control Cantonese sausage was obtained. Samples were inoculated with S. condimenti (SC)and M. caseolyticus (MC)at 106cfu/g,respectively. After drying, the products were vacuum-packaged and stored at –20Cuntil analysis.
Isolation and Identificationof Bacteria
Staphylococci were isolated from Cantonese sausages. The strains were selected by standard microbiological methods using appropriate dilutions in buffered peptone solution (Simonováet al . 2006). Dilutions were plated onto mannitol salt agar plates and incubated at 37C for 48h. After incubation, the colony forming units were counted. Among gram-and catalase-positive strains, 64belonged to the staphylococci group (64strains). According to the sensory evaluation, four strains producing aroma were selected for further identificationand testing. They were further characterized by the following complementary biochemical tests:tolerance to NaCl and nitrite, acid produc-tion, adhesion assay, and growth ability, lipolytic activity and proteolysis activity by the methods of Papamanoli et al . (2002),Rantsiou et al . (2005)and Simonováet al . (2006).Two strains were confirmedsuitable to be starter cultures in Cantonese sausages. They were further identifiedby the means of polymerase chain reaction-denaturing gradient gel electrophoresis described by Rantsiou et al . (2005).
Analysis of Volatile Compounds
SPME Fibers. V olatile compounds were extracted by SPME method. An SPME device (Supelco,Bellefone, PA) was used in this study, with 50m m of divinylbenzene/carboxen/polydimethylsiloxanefiber.Before the analysis, the fiberwas preconditioned in the injection port of GC as indicated by the manufacturer.
Procedure
For each experiment, 5g of Cantonese sausage was minced and weighed into a 50-mL headspace vial and sealed with a Teflonsilicone septum. The vial
EFFECTS OF S. CONDIMENTI AND M. CASEOLYTICUS ON CANTONESE SAUSAGE 847was left at 30C in a thermal block for 1h to equilibrate the headspace. Then, an SPME fiberwas exposed to the headspace while maintaining the sample for 60min. The compounds absorbed by the fiberwere identifiedand quantifiedby GC–MS.
Identificationand Quantificationof Volatile Compounds
The flavorcompounds adsorbed by the fiberwere desorbed in the injec-tion port of GC–MS(FinniganMAT 8230GC-MS, Palo Alto, CA) for 3min at 230C with a splitless injection mode. The compounds were separated in a DB-1capillary column (30m ¥0.25mm internal diameter, 1m m of filmthickness). Helium was used as carrier gas with a linear velocity of 1mL/min.The GC oven temperature program was started when the fiberwas inserted and held at 50C for 3min, ramped to 120C at 5C/min,holding for 2min, and then to 220C at 10C/min,holding for 10min. The transfer line to the mass spec-trometer was maintained at 230C. The mass spectra were obtained using a mass selective detector by electronic impact at 70eV . The detector voltage was 350V , and the scan range of m/zwas 35–395amu.
Identificationof flavorcompounds was carried out according to the reten-tion time and by comparing mass spectrum data (RT/MS)of the samples with the the National Institute of Standards and Technology literature database. V olatile compounds were quantifiedby calculating the peak area.
RESULTS AND DISCUSSION
Identificationand Characterization of Strains
The DNA sequences were analyzed. The comparison for DNA similarity was performed with the GenBank, European Molecular Biology Laboratory, DNA Databank of Japan and Protein Data Bank database (Blaiottaet al . 2003). Through this analysis, two strains were identifiedas S. condimenti and M. caseolyticus , respectively.
Analysis of Volatile Compounds
The three samples had 22, 22and 25volatile compounds, respectively. Compounds identifiedin the samples are listed in Tables 1–3.Their origins are also listed. Four hydrocarbons, two alcohols, fivealdehydes, one ketones, eight esters and two heterocyclic compounds were identifiedin control; two alcohols, six aldehydes, one ketones, 10esters and three heterocyclic com-pounds were found in SC; one hydrocarbon, four alcohols, seven aldehydes, three ketones, eight esters and three heterocyclic compounds were observed in
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TABLE 1.
VOLATILE COMPOUNDS IDENTIFIED IN CANTONESE CONTROL SAUSAGES AND
THEIR ORIGINS
Number IdentificationCompound
Ethanol
1-octen-3-ol
Pentanal
Hexanal
Heptanal
2-octenal
Nonanal
2,3-octanedione
Propanoic acid, ethyl ester
1-butanol, 3-methyl-, formate
Butanoic acid, ethyl ester
Propanoic acid, 2-hydroxy, ethyl ester
Butanoic acid, 3-methyl-, ethyl ester
Pentanoic acid, ethyl ester
Hexanoic acid, ethyl ester
Octanoic acid, ethyl ester Area (%)47.990.900.745.150.350.231.281.660.492.831.451.750.460.234.361.05
5.52
5.49
0.14
0.10
10.70
0.21Origin Wine Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Microbial Microbial Microbial Microbial Microbial Microbial Microbial Microbial Unknown Lipid Unknown Unknown Unknown Contaminant Alcohols (48.89%)1RT/MS2RT/MSAldehydes (7.75%)3RT/MS4RT/MS5RT/MS6RT/MS7RT/MSKetones (1.66%)8RT/MSEsters (12.62%)9RT/MS10RT/MS11RT/MS12RT/MS13RT/MS14RT/MS15RT/MS16RT/MSAlkanes and alkenes (11.25%)17RT/MSMethylene chloride 18RT/MSHexane 19RT/MSn-ethyl-1,3-dithioisoindoline 20RT/MSn-ethyl-1,3-dithioisoindoline Heterocyclic compounds (10.91%)21RT/MSBenzene 22RT/MSToluene
RT/MS,retention time and mass spectrum data.
MC. Cantonese sausages inoculated with starter cultures had higher alcohols, aldehydes, ketones and esters as well as lower hydrocarbon and cyclic com-pounds than the control.
SC and MC showed higher levels of alcohols than the control, especially for 2-pentanol and 2-(2-butoxyethoxy)-ethanol,including ethanol and 1-octen-3-ol in MC. This result could be attributed to the starter culture-related fermentation and lipid oxidation. The results showed that ethanol was the most abundant desorbed volatile compound. In the control, 47.99%of ethanols were determined, while in SC and MC, 53.00and 46.16%of ethanols were
EFFECTS OF S. CONDIMENTI AND M. CASEOLYTICUS ON CANTONESE SAUSAGE 849
TABLE 2.
VOLATILE COMPOUNDS IDENTIFIED IN CANTONESE STAPHYLOCOCCUS CONDIMENTI
SAUSAGES AND THEIR ORIGINS
Number IdentificationCompound
Ethanol
1-octen-3-ol
Pentanal
Hexanal
Heptanal
2-heptenal
2-octenal
Nonanal
2,3-octanedione
Acetic acid, ethyl ester
Propanoic acid, ethyl ester
1-butanol, 3-methyl-, formate
butanoic acid, ethyl ester
Ethyl 2-hydroxy-propanoate
Butanoic acid, 2-methyl-, ethyl ester
Butanoic acid, 3-methyl-, ethyl ester
Pentanoic acid, ethyl ester
Hexanoic acid, ethyl ester
Octanoic acid, ethyl ester Area (%)53.001.440.888.640.430.590.471.442.733.270.503.232.172.490.230.630.324.791.22
4.61
0.22
0.10Origin Wine Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Fermentation Microbial Microbial Microbial Fermentation Microbial Microbial Microbial Microbial Microbial Unknown Contaminant Unknown Alcohols (54.44%)1RT/MS2RT/MSAldehydes (12.45%)3RT/MS4RT/MS5RT/MS6RT/MS7RT/MS8RT/MSKetones (2.73%)9838Esters (18.85%)10RT/MS11RT/MS12RT/MS13RT/MS14RT/MS15RT/MS16RT/MS17RT/MS18RT/MS19RT/MSHeterocyclic compounds (4.81%)20RT/MSBenzene 21RT/MSToluene 22RT/MS1,2-dihydro-1,4-diphenylphtyalazine
RT/MS,retention time and mass spectrum data.
identified,respectively. A similar result was reported by Du and Ahn (2001).These authors also suggested that wine is responsible for the development of flavorin Cantonese sausages. A large amount of wine (approximately4%in comparison with the weight of meat) is usually added at the beginning of the sausage-making process. This leads to a higher proportion of ethanol and other volatile compounds in the sausage.
Many researchers have studied the role of starter culture in the production of aldehydes and have suggested that species of the genus Staphylococcus are more important than Lactobacillus and Pediococcus in relation to the produc-tion of volatile compounds (Berdaguet al . 1993). From the data in Tables 1–3,aldehydes account for 7.75, 12.45and 13.1%of the total volatile compounds
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TABLE 3.
VOLATILE COMPOUNDS IDENTIFIED IN CANTONESE MICROCOCCUS CASEOLYTICUS
SAUSAGES AND THEIR ORIGINS
Number IdentificationCompound
Ethanol
2-pentanol
1-octen-3-ol
2-(2-butoxyethoxy)-ethanol
Pentanal
Hexanal
Heptanal
2-octenal
Nonanal
2-nonenal
6-(hydroxyl-phenyl-methyl)-2,2-dimethyl-
Cyclohexanone
1-octen-3-one
2,3-octanedione
Acetic acid, ethyl ester
Butanoic acid, ethyl ester
Ethyl 2-hydroxy-propanoate
Butanoic acid, 2-methyl-, ethyl ester
Butanoic acid, 3-methyl-, ethyl ester
Pentanoic acid, ethyl ester
Hexanoic acid, ethyl ester
Octanoic acid, ethyl ester Area (%)46.163.471.563.631.088.530.420.511.820.150.780.142.732.752.232.570.210.540.336.602.20
0.15
3.33
0.18
0.18Origin Wine Fermentation Lipid oxidation Fermentation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Lipid oxidation Alcohols (54.82%)1RT/MS2RT/MS3RT/MS4RT/MSAldehydes (13.1%)5RT/MS6RT/MS7RT/MS8RT/MS9RT/MS10RT/MSKetones (3.65%)11RT/MS1213RT/MSRT/MSLipid oxidation Fermentation Microbial Fermentation Microbial Microbial Microbial Microbial Microbial Fermentation Unknown Contaminant Unknown Esters (17.43%)14RT/MS15RT/MS16RT/MS17RT/MS18RT/MS19RT/MS20RT/MS21RT/MSAlkanes and alkenes (0.15%)22RT/MSL-limonene Heterocyclic compounds (3.69%)23RT/MSBenzene 24RT/MSToluene 25RT/MS2-pentyl-furan
RT/MS,retention time and mass spectrum data.
in the control, SC and MC sausages, respectively. Aldehydes in Cantonese sausages were linear aliphatic from C 4up to C 10. Among these, hexanal and nonanal were the main aldehydes found in Cantonese sausage. This result is similar to that reported by Meynier et al . (1999).Hexanal has been suggested as the oxidation product of linoleic acid and the detection of hexanal indicates the degree of lipid oxidation in the sausage (Duand Ahn 2001). SC and MC
EFFECTS OF S. CONDIMENTI AND M. CASEOLYTICUS ON CANTONESE SAUSAGE 851sausages showed higher levels of aldehydes than the control, especially for hexanal and nonanal. These aldehydes are produced during the oxidative degradation of unsaturated fatty acids (Mottram1998). After inoculating the two strains, the oxidative degradation of unsaturated fatty acids increased because of the lipolytic activity of S. condimenti and M. caseolyticus . Besides aldehydes, there are many other volatile compounds arising through lipid oxidation, such as hydrocarbons, alcohols and ketones. They were present in small proportions compared with aldehydes. SC and MC sausages had larger amounts of hydrocarbons, alcohols and ketones.
As a whole, volatile compounds generated by lipid oxidation accounted for 10.31%of the total volatile compounds of control sausages, which was lower than those for the samples inoculated with starter cultures (16.62%in SC and 23.85%in MC). These results are consistent with the biochemical char-acteristics of S. condimenti and M. caseolyticus , which possess lipolytic activ-ity and the function of oxidative deamination and transamination (Klooset al . 1998; Probst et al . 1998).
Esters are another group of volatile compounds that showed differences between the control and the sausages inoculated with S. condimenti and M. caseolyticus . In control, SC and MC sausages, 8, 10and 8esters were identified,which accounted for 12.62, 18.85and 17.43%of total volatile compounds, respectively (Tables1–3).The results indicate that S. condimenti and M. caseolyticus could increase the amount of ethyl esters, most of which resulted from the microbial esterificationof acids (derivedfrom sugar fermen-tation, lipolytic activity or degradation and oxidation of amino acid) with alcohols. Our result is in agreement with those previously obtained in dry sausages by Stahnke (1994,1995). Montel et al . (1996)have also found that S. xylosus and S. carnosus are the main producers of ethyl esters, giving rise to sausage models with a better flavor.
In the present study, sausages inoculated with S. condimenti and M. caseolyticus showed higher levels of 3-methyl ethyl butanoate, 2-methyl ethyl butanoate and ethyl acetate than control sausages. This fact might be attributed to the inoculation of Staphylococcus species, which can transform branched amino acids (valine,isoleucine and leucine) during sausage ripening into 3-methy butanal and 2-methyl butanal through a nonenzymatic Strecker reaction (Barbieriet al . 1992; Berdagu et al . 1993) or by microorganism metabolism (Meynieret al . 1999). Then the corresponding alcohols were formed by the reduction of the aldehydes. Or the aldehydes are oxidized to the corresponding acids and esterifiedwith ethanol by Staphylococcus , producing 3-methyl ethyl butanoate and 2-methyl ethyl butanoate (Stahnke1994; Meynier et al . 1999; Hérranzet al . 2005).
Greater amounts of ethyl esters in SC and MC sausages can be consi-dered favorable because they are essential to the overall flavorof fermented
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sausages, providing fruity notes or masking rancid odor (Hérranzet al . 2005). Compared with the volatile compounds derived from the lipid degradation and oxidation, esters have a lower proportion in Cantonese sausages, but they play a more important role in the flavorof Cantonese sausages because of their very low threshold value. From the results shown, we inferred that esters are important for the flavorof Cantonese sausages, and 2-hydroxy ethyl pro-panoate is responsible for the unique flavorof Cantonese sausages.
In the present study, benzene and methyl benzene were found in the three samples. Methylene chloride and n-ethyl-1,3-dithioisoindoline were found in control sausages. These volatile compounds did not directly originate from the meat products. Similar results were reported by Du and Ahn (2001).These authors detected benzene, methyl benzene and chloroform in Cantonese sausages, and they pointed out that these volatile compounds are from the packaging materials. Further work will be carried out in order to interpret this phenomenon.
In addition, L-limonene was found in MC. It is probably formed through the extensive degradation of lipids during the ripening of Cantonese sausages after inoculation with M. caseolyticus . 2-pentyl-furan was also found in MC because of the autoxidation caused by M. caseolyticus .
CONCLUSION
From the results of this work, the use of S. condimenti and M. caseolyti-cus is beneficialto the degradation and oxidation of lipid and amino acids. S. condimenti and M. caseolyticus could improve the overall flavorof Can-tonese sausages because of an increase in the generation of volatile com-pounds. The study also indicates that S. condimenti and M. caseolyticus have good potential to be starter cultures in sausages. Unlike in previous reports, branched aldehydes were not detected in the three samples used in the present study. Branched aldehydes may have been converted into their corresponding esters by transamination, oxidative deamination and microbial esterification.This study indicates that the majority of volatile compounds from Cantonese sausages are alcohol. Although volatile compounds generated from fatty acids, amino acids, microorganism and fermentation only account for a small pro-portion, they play an important role in the flavorof Cantonese sausages.
ACKNOWLEDGMENTS
This work was financiallysupported by the Science and Technology Foundation of Guangdong Province (GrantNo. 2005A20303002) and the
EFFECTS OF S. CONDIMENTI AND M. CASEOLYTICUS ON CANTONESE SAUSAGE 853University-Enterprise Partnership Foundation of Guangdong Province (GrantNo. 2006D90202001). The authors want to acknowledge Yufen Chen and Caizhen Yang for their technical help.
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