Microbiological safety of traditional and starter-mediated processes for the manufacture of Italian dry sausage

lnternattonal Journal ofFoodMtcrobtology, 7 (1988) 49-62 Elsevier

49

JFM00221

Microbiological safety of traditional and starter-mediated processes for the manufacture of Italian dry sausage R.A. Holley 1, Anna M. Lammerding 2 and F. Tittiger 3 1 Food Research Centre. Agrtculture Canada. Ottawa, Ontarto, Canada. " A m m a l Patholog} Laboratory. A grtculture Canada, Guelph. Ontario, Canada and ~ Meat H~ gtene Dtvtston, Agriculture Canada. Ottawa, Ontarw, Canada (Recewed 26 Februar~ 1988, accepted 19 April 1988)

M~crobmloglcal changes occurnng during the commerctal manufacture of Itahan dry sausages (Genoa and salamettl) were studted m two urban Canadtan centres over a 5 month penod A companson was made between 6 plants which used bacterial starter cultures and 4 plants where more tradmonal processes (without starters) were used A total of 600 samples of raw, fermented and fimsbed products were tested for the presence of cohforms, salmonellae, staphylococct, streptococcL the rate of pH reductton and final water activity (a w) Numbers of total bacteria peaked earher and were slgmficantly higher m sausages at the fermentauon stage produced with starter cultures than m those tradmonally manufactured. This corresponded with a more rapid drop m pH of the starter-moculated products Staphylococct and streptococc~ were stgmficantly higher m starter-fermented Genoa sausages at the fermentauon stage, but no stgmficant differences were seen m the rmcrob~olog~cal content or a,~ of mature fimshed sausages manufactured by the two different techmques Of 128 randomly chosen ~solates of coagulase-pos~twe staphylococcu 34.4% were enterotoxm producers and 80% of these produced type A toxin Enterotoxagenlc staphylococc~ were found m 2 different samples of fimshed salamettt and one sample of fimshed Genoa made w~th starter cultures and m one sample of fimshed Genoa made v,athout added culture. Total numbers of staphylococci m these samples were not > 500/g No correlation between the method of manufacture and presence of enterotoxagemc staphylococc~ could be made Fwe subsamples from one lot of raw Genoa were the only samples posmve for Salmonella d u n n g this study Results indicated that low temperature tradmonal fermentatmns can y~eld products which are as safe as those produced by the higher temperature starter-controlled process One of the most tmportant elements m the traditional process was beheved to be the selection and use of raw matertals of the highest posstble quahty

Key words" Starter culture, Lacttc Bacteria, Meat fermentauon, Sausage, dry., Staphylococcus aureus

Correspondence address (also present address) R A Slmcoe St, London, Ontario N6A 4M3, Canada

Holley, Techmcal Services, John Labatt Ltd

0168-1605/88/$03.50 ~ 1988 Elsevier Science Pubhshers B V (Btomedlcal Dt~aslon)

150

50 Introduction During the last two decades many studies have been conducted to answer questions concerning the microbial safety of processes used to prepare uncooked dry sausages suitable for ethmc needs. Organisms of primary pubhc health concern are Staphylococcus aureus and to a lesser extent Salmonella spp. winch regularly contaminate raw materials and processing equipment. It is doubtful that manufacturing processes currently in use will elmunate these pathogens completely ~f raw matermls are heawly contan~nated, (Goepfert and Chung, 1970; Metaxopoulos et al., 1981). The use of first quahty meats in conjuncuon w~th starter culture bacteria, plus adoption of processing conditions winch nnnlmlze ripening times at temperatures above 15 6 ° C are critical points for pathogen control. At the same t~me, a sufficient interval for bacterml fermentation to reduce the initial p H of the meat mix to 5 3 (Bacus and Brown, 1985a, b), must be provided These conditions will yield safe and acceptable uncooked products Bacterial cultures available (usually lactobacilh, pedlococcl, M~crococcaceae or mixtures of these) are used m formulations where either a rapid fermentation (8 h at 38 ° C) or somewhat slower fermentations yielding less acidity (48 h at 24 ° C) are desired (Smith and Palumbo, 1983: Bacus, 1984, Bacus and Brown, 1985a, b, H a m m e s et al., 1985a, b). The chemical acldulant glucono-8-1actone (GdL) has also been used with some success e~ther alone or w~th starter cultures for rapid acid production (Winter, 1986). However, in sausages fermented at low temperature ( _< 20 ° C) use of G d L ts considered detrimental to flavour and accelerates development of rancidity (Lucke and Hechelman, 1986). Rapid acidification by G d L or bacterial cultures m products which are fermented and ripened at temperatures of _< 20 ° C for more than 3 - 4 weeks ts considered by some to be unnecessary (Lucke and Hechelman, 1986; Slmonettl et al.. 1983) although the value of starters in standard~zing the process is recognized. In those products which are intended to have a very long shelf-hfe (e.g., G e n o a salanu) rapid production of acid tends to retard development of the acid-unstable m~croflora (catalase positive Micrococcaceae) responsible for development of cured color and prevention of premature fat rancidity (Lucke and Hechelman, 1986: Holley et al., 1988a). In low-temperature fermented sausages, temperature and water activity ( a , , ) replace rapid acidulation as the primary 'hurdles' to prevent proliferation of bacterial pathogens (Leistner, 1985). During the slow rapening process, micrococci initially dominate the microflora for up to 4 days and thereafter they are overtaken by growth of lactobacilli (Sxmonetti et al.. 1983: Bacus, 1984) Eventually. oxidation of amino acids and lactic acid by yeasts and mold can rinse the meat p H to levels approximating that of freshly slaughtered animals (Lucke and Hechelman, 1986). Although growth by staphylococci has been noted under certain circumstances to occur at < 15 ° C (Troller, 1986; Palumbo, 1986) the threat from these orgamsms is greatly reduced at low sausage fermentation temperatures (Bacus, 1984). Indeed, Salmonella is recognized as a greater potential hazard in these products because of ~ts greater of its greater abihty to compete and grow at temperatures _< 1 0 ° C

51 (Palumbo, 1986). Additionally, Salmonella was able to survive reduced a,, better at refrigerator temperatures (Goepfert and Chung, 1970: Holley, 1985). The present work was undertaken to examine the microbial safety of currently used procedures in Canada for the manufacture of Italian dry sausages by both local traditional (low temperature) and modern (starter culture-mediated) procedures.

Methods Ten commercial meat plants registered with Agriculture Canada as producing raw dry cured Italian-type sausages were selected for the study. Five plants were located m Montreal (plants A to D and J), while the remainder (plants E to I) operated in the Toronto area. Two types of sausages were studied: salametn which wmghed about 500 g and Genoa which weighed about 2.5 kg. Each plant manufactured both types of sausage according to the same formulation with rmnor variations from plant to plant. Conditions of manufacture (Tables I and II) were not altered for the purpose of th~s study. Six plants used commercial bacterml starter cultures and pe&ococci were common (but not exclusive) to them all. Sodium nitrate ( < 200 ppm) was used with nitnte ( < 200 ppm) in all formulations at 9 plants. At plant F, where a starter was used that contained only Pe&ococcus, mtr~te (156 ppm) alone was used. None of these sausages was smoked. When starter cultures were used, sausages were formulated with a (mean +_ SD) NaCI content of 3.1 +_ 0.4%, (w/w), whereas a slightly htgher level of salt was used by all traditional producers of sausages (3.3 + 0% w/w). At three plants (A, D and F) sausages were formulated with < 0.2% ( w / w ) monosodmm glutamate (MSG), whereas erythorbate ( _< 0 1% w / w ) was used at all plants where starter cultures were used. None of the traditional manufacturers formulated products with MSG and only two of these used ascorbate or erythorbate (I and J). The acidulant G d L was not used at any of the 10 plants surveyed. Starter culture-inoculated meat mixtures containing spices and cure were stuffed into casings at < 4 ° C , held at 1 0 ° C for about 8 h, moved into the 'green' or fermentation room at 2 4 - 3 2 ° C for 24-48 h at l'ugh relative humadity (98% RH). Gradually, temperature and R H were reduced to about 1 8 ° C and 90%, respectively, until a pH of just over 5.0 was reached (usually 3-5 days). Sausages were then moved to drying rooms where further gradual reductions in temperature and RH were made 12-7 o C and 75-65% RH over a period of one to two months depending on sausage diameter (Table I). When manufactured traditionally, ground pork ( < 4 ° C) was spiced, the cure-salt mixture added and the batter was either stuffed in casings and held at 4 ° C for 1-3 days or held m 250-500 kg lots in stainless steel vats for 2 - 7 days (usually 3 days) at < 7 ° C (usually 4°C). There was no evidence of 'backsloppmg' (adding mature sausage to the mixture) at any of these plants. After being held in vats, the meat-cure mixture was stuffed into casings, held either briefly ( < 24 h) at elevated temperature ( 2 7 - 3 4 ° C and 98%, RH) or held for as long as a week at 2 1 - 2 4 ° C at q

I

acrdrlactrcr

was not used at plant F, but was present m all other plant formulations SO-90 mm dmmeter) except plant B where natural casmgs were used h Relative hunu&ty

8 Nltrate

All plants used regenerated

collagen

(salamettl.

55-65

mm: Genoa,

13OC, 60 d, 17% RH 15-lo C, 30-60 d, 70% RH 18-13°C. 45 d, 75-658 RH 18-12°C, 25-94d. 84-71s RH 12 o C. 40-60 d, 70% RH IO o C, 28-50 d, 70-60% RH

Dvng

casmgs

Pedrocoma

26°C,to48h,98%RH h topH53 30 o C, 24-30 h, 90% RH to pH 5 3 24OC, 24 h, 18”C, 5 d. 85% RH to pH 5 1 25-2S°C, 24 h,98% RH, 18°C. 84% RH to pH 5 3 29°C,24h,90%RHtopH53 32O C, to 48 h, 85% RH to pH 5 0

’

+ + + + + +

Fermentation

When nuxtures used, beef was added to about 33% (Plant B), 18% (Plant C), 15% (Plant E) C = conunerclal; 0 = own formulation A nuxture of pedmcocc~ (2 types), L. planfanm and M cmrrans, accordmg to manufacturer’s speclflcatlons An equal mxture of Lactacel 75 (Pedmocw) and Lactacel 444 (M~crococcus), accordmg to commercial speclflcatlons Sfaphylococcus c~mosus and Pedmcoccus penrosoceus, accordmg to commercial speclflcatlons

+ + -

0 0 C C c 0s

+ + + -

+ + + + + +

b

cultures

’ ’ ’ d ’

Rosellac-AC Rosellac-A Rosellac-A Lactacel d Trumark LT-IIM ’ Trumark F-100 ’

A B C D E F

Cure Frozen

‘plce

Fresh

Beef

with starter

Meats a

dry sausages

Pork

of raw. fermented

Starter

manufacture

Plant

Commercial

TABLE

53 TABL E II T r a d m o n a l a manufacture of raw fermented dry sausages wtthout starter cultures Plant

G

Pork b

Cure

Pre-

Fresh

Frozen

Sptce c

fermentation d

+

+

C

+

H I J

+

Fermentation

Drying

2°C, 4d

2 7 - 3 4 ° C , 24 h

C

4°C, 2-7 d

1 7 ° C , 24 h

-

O

< 7 ° C , 2 d. vat

12°C, 7 d

+

O

< 7 ° C , 1-2 d, vat

24-21°C, 7 d

1 6 - 1 2 ° C, 25-40 d, 75-70% RH e 1 6 - 1 0 ° C, 25-45 d, 70% RH 1 2 - 9 ° C 20-45 d, 79% R H 1 6 ° C , 30 d. 77% RH, 1 2 ° C , 30 d. 55% RH

Italian style, no starter cultures b Pork only C = commercial, O = own formulation, nitrate present in all formulations d Mtxture was either stuffed in casings and refrigerated or was held m 250-500 kg lots in vats prior to fermentation At plant I, after stuffing, sausages were held 18 h at 4 ° C before fermentation e Relative huttu&ty

a

low R H (75%). In a single plant (I) the temperature after stuffing was maintained at about 1 2 ° C for a week. Drying at these plants was usually conducted m two stages of approximately equal length (16 ° C and 77% R H followed by 1 2 ° C and 70%, RH). Each stage could last for 12-30 days depending on sausage diameter and the desired dryness (Table II).

Sausage samphng Samples of salametti and G e n o a sausages were taken from commercial production lots by Agriculture Canada Inspection staff at each of the 10 plants, and refrigerated for a m a x i m u m of 2 days before being analyzed. Raw product samples (each consisting of 5 subsamples of 100 g each) were taken after meat gnndmg, but before additmn of salt, binder, starter cultures or cure ingredients. Fermented product samples (5 subsamples of 100 g each, or in the case of G e n o a sausage, one entire sausage) were taken after the product left the 'green' room before initiation of drying. In those instances where starter cultures were not used, the fermented samples were taken 7 days after the initiation of fermentation. Finished product samples (taken as noted for fermented meat) were obtained when the product was released for sale. Samples were analyzed at the nearest of two private laboratories, one in Toronto, the other in the Montreal area. Each type of product was sampled twice at each plant with at least a 3 week interval between sampling of identical product at the same stage of maturation All subsamples were taken from the same production lot, although samples of raw, fermented and finished products may not have been taken sequentially from identical lots at each plant. A total of 600 subsamples were analyzed d u n n g this

54 study. Before analysis, sausage casings were carefully removed and a vertical slice of 11 g was taken from the circumference to the centre in a wedge-shaped configuration to yield about one third of the sausage circumference/subsample To each subsample of 11 g, 99 ml sterile 0.1% ( w / v ) peptone was added and massaged for 30-90 s in a Stomacher 400 (A.J. Seward, Blackfriars Rd., London, U.K.). The homogenate was decimally diluted with peptone water as deemed appropriate for microbiological analysis. Microbial enumeration and identification

Methods followed were essentially those considered as 'acceptable" methods by the Health Protection Branch, (HPB), Health and Welfare, Canada (1983). Total numbers of aerobic bacteria were determined according to method M F HPB 18 which consisted of pour plating duplicate ahquots of sample homogenate with Standard Plate Count Agar followed by incubation at 35 ° C for 48 h. Tests for cohforms were performed on each subsample after HPB method MF HPB 19. A 3 and 5 tube Lauryl Tryptose Broth most probable numbers technique was used. Four to 6 dilutions were tested, depending on sample age and tubes were incubated at 35 ° C for 24-48 h. Aliquots from each p o s m v e tube (growth and gas) were transferred to Brilliant Green Bile Broth (2% BGLB) for coliform confirmation and to EC Broth for detection of fecal cohforms. Broths were incubated for 48 h at 35 ° C and 44.5 o C, respectively. Tubes of EC Broth showing growth were streaked on Eosin Methylene Blue (EMB) Agar plates and incubated at 35 ° C for 24 h. Each presumptive colony was confirmed as Eschertchta coh If it exhibited a positive indole and a negative citrate reaction. The MF HPB 21 method was used to determine numbers of Staphylococcus aureus present in subsamples. Prepared Baird-Parker Agar plates were spread In duplicate with 0.2 ml of appropriate sample dilutions and incubated at 35 ° C for 48 h. Five presumptive colonies from each countable plate were transferred to Tryptic Soy Agar (TSA) plates and tested for coagulase activity. Colonies showing typical appearance and coagulase activity were recorded as S aureus Each of the two private laboratories sent approximately 100 such isolates to the Animal Pathology Laboratory, Food Production and Inspection Branch, Agriculture Canada, Guelph, Ontario, Canada, for examination of enterotoxm production. At the Guelph laboratory isolates were tested for either catalase or thermostable nuclease (TNase) activities and subjected to a reverse passive latex agglutination assay for enterotoxms A, B, C and D (Denka Seiken Ltd., Japan, available from Oxoid Canada Inc., Nepean, Ontario, Canada). Enterococci were enumerated in duplicate using pour plates of K F Streptococcus Agar to which 10 ml/1 1% ( w / v ) aqueous triphenyltetrazohum chloride (TTC) was added. Plates were incubated at 35 or 4 3 ° C for 48 h and all red or pink colonies were counted. Detection of Salmonella (MF HPB 20) was performed by addition of 25 g of sample to 225 ml Nutrient Broth followed by incubation at 35 ° C for 18-24 h. 1 ml of this pre-enrlchment was transferred to 9 ml selenite cystine and tetrathionate

55 broths which were incubated at 35 ° C and 43 ° C, respectively, for 24 h. Broths were streaked on Bnllian Green Sulfa Agar (BGS) and Bismuth Sulfite Agar (BS) and incubated at 3 5 ° C for 24-48 h. Presumptive posmve colonies were transferred to Triple Sugar Iron Agar (TSI), I-ysme Iron Agar (LI) and Urea and Lysme Decarboxylase Broths which were incubated at 3 5 ° C for 24 h. Somatic O antisera (Difco) and API 20E strips (API Laboratory Products, St. Laurent, Quebec, Canada) were used for confirmation.

Measurement of pH Fisher Accumet p H meters (Fisher Sci. Ltd., Toronto, Ontario, Canada) were used to determine sausage pH. Electrodes were e~ther inserted d~rectly into 3 g aliquots of ground meat or measurements were made using 10 g of subsample homogenized with 15 ml of sterile distilled water.

Water

aCtlUllF

Water actwlty ( a ~ ) was determined with a N o v a s m a thermoconstanter TH-2 (Zurich, Switzerland) a,,, meter standardized at 0.901 and 0.529 (25°C) following eqmhbratlon for at least one hour and values corrected to 20 °C. The Montreal laboratory measured aw with a Lufft model ~ 5 8 0 3 meter (G. Lufft G m b H & C o . , Stuttgart, F.R.G.) at 2 0 ° C .

Data anal),sls Arithmetic means of viable bacteria, p H and aw were calculated for each of 5 replicate analyses per sample for each type of sausage. The process was repeated for the complete second experimental set and the resulting paired means were grouped according to type of sausage, stage of production and type of process used (starter culture or tradmonal). Six pairs of means where starters were used compared with 4 pairs of means generated by use of the traditional process in an analysis of variance procedure (VMS version, SAS release 5.03, C P U I D model 8600, SAS Institute Inc., Cary, NC, U.S.A.).

Results

Sausage analyses N o significant differences were noted among the numbers and types of bacteria present m raw and finished salametti and G e n o a sausages when formulated with or without starter cultures (Table III). There were also no differences in initial and final p H values or final aw which could be related to the method of manufacture. In both salametti and Genoa sausages after the fermentation stage the numbers of total bacteria present were significantly ( P < 0.01) higher in starter-inoculated products.

56 TABLE III M~crob~ologlcal comparison a between starter-aodulated and naturally fermented sausages Bacterial

Raw

type

Starter

Traditional Starter

Traditional

558_+103 136_+058 265_+116 181_+165 257_+097

534_+086 109_+059 223_+068 113_+090 330+_105

624_+165 ~ 769 113+063 175 170_+127 014 003_+074 -050 263+169 364

Salametti Totalnumbers Staphylococci Cohform~ E ~oh Streptococo pH av,

Fermented

842_+090' 219_+152 129_+107 064_+114 388+126

Finished

618-+013 615-+011 4.80__+021 ~ 570__+038 ~ . . . .

Starter

Tradmonal _+081 +119 _+101 +027 _+132

504 +047 0837_+0048

820 122 -002 +065 362

_+052 _+086 +084 _+009 _+200

505 -+017 0848-+0042

Genoa

Totalnumbers Staphylococci Cohforms E cob Streptococci

554-+132 169-+066 256-+099 203-+1.40 284-+085

534_+109 134_+091 238_+063 088_+119 261-+078

pH a~

618-+020 633_+046 -

825_+093 ~ 600_+165 ~ 778 206_+101 b 103_+063 b 158 088_+120 155+095 -006 052_+113 008_+12 -035 399+136 b 254+090 b 360 473_+015 ~ 579_+029' -

_+094 -+074 _+079 +052 -+163

488 _+041 0838_+0042

814 101 014 -065 3.74

-+053 _+044 -+089 _+009 _+246

475 -+019 0882_+0052

Mean_+SD of lOgl0 CFU/g meat For pH and a,,, mean_+ SD of all observauons b Numbers in same roy, are slgmflcantly different with P < 0 05 Numbers in same row are slgmficantly different ~tth P < 001

Similarly, the p H of s t a r t e r - i n o c u l a t e d p r o d u c t s was significantly ( P < 0.01) lower than that of t rad it io n a l sausages. Interestingly, n u m b e r s of b o t h s t r e p t o c o c c i an d coagulase positive s ta p h y l o c o c c i were significantly higher ( P < 0 . 0 5 ) in G e n o a sausages c o n t a i n i n g a d d e d starter cultures than in the t r a d i t i o n a l l y m a n u f a c t u r e d products. A l t h o u g h there was no significant d i f f e r e n c e b e t w e e n m e a n n u m b e r s of b o t h these types of bacteria in f e r m e n t e d salamettl, their n u m b e r s were higher in t r e a t m e n t s where starter cultures were used.

Enterotoxlgentc staph.vlococcl A total of 128 coagulase positive s t a p h y lo co cci cultures f r o m 8 plants were e x a m i n e d for e n t e r o t o x m p r o d u c t i o n by reverse passive agglutination. O f these, 44 cultures (34.4%) were e n t e r o t o x l n producers. All but o n e of 32 e n t e r o t o x i n producers tested p r o d u c e d thermonuclease. Enterotoxans of m u l t i p l e types were detected in 37 of the cultures. O f the remainder, 6 p r o d u c e d type A an d o n e p r o d u c e d type B only. 80% of cultures p r o d u c e d type A, 57% p r o d u c e d type D, 39% p r o d u c e d type C and 32% p r o d u c e d type B e n t e r o t o x i n Toxagenic s t a p h y l o c o c c i were f o u n d in b o t h G e n o a a n d salametti sausages at all stages of p r o d u c t i o n . A c o r r e l a t i o n c o u l d not be d r a w n b e t w e e n the presence of e n t e r o t o x i g e n t c s t a p h y l o c o c c i and p r o d u c t i o n m e t h o d since e n t e r o t o x i n testing was not d o n e on isolates f r o m all

57 plants and equal numbers of isolates were not tested from each of the remamlng plants. Among those samples which contained toxlgenic staphylococci, total staphylococci were below 1000/g meat except for 3 samples of fermented salametti ( < 1800/g) and 2 samples of raw G e n o a (7800/g). Enterotoxigemc staphylococm were found in 9 subsamples of finished salametti which represented 2 different samples and both were from plant A. These samples contained 500 staphylococc~/g meat. Toxagenic staphylococci were also found in 2 samples of fimshed G e n o a sausage. One sample was from plant E, the other was from plant I and these samples contained a total of 300 and 100 staphylococci/g, respectively. Salmonella Five subsamples from one lot of raw meat for G e n o a manufacture at plant I were found to be contaminated with Salmonellae. Tins lot of sausage was retested when fully mature and was negative for Salmonella. N o other samples taken during this study were positive for Salmonella.

Discussion Three procedures have been used by manufacturers to ensure that an active culture of lactic bacteria is present in raw meat formulated for dry sausage production. Culture enrichment or aging of salted (2-4% NaC1) meat at low temperature (e.g., 4.4°C for up to 10 days) has been shown to yield increased numbers of lactic acid b a c t e n a (Raccach and Baker, 1979; Bourgeois et al., 1983), although there is a report to the contrary (Deibel et al., 1961). Masters et al., (1981) found that exposure of meat to 4 . 4 ° C for 48 h did prove to be more restrictive to growth of added Salmonella than when meat was not pre-incubated at low temperature and fermented without bacterial starters. Culture enrichment is one of the older techniques used for dry sausage manufacture and was used m 4 of the 10 plants studied (Table II) where starter cultures were not used. The second method involves ' b a c k inoculation' or addition of a mature sausage(s) to the raw meat formulation and in tins way provides an lnoculum of organisms capable of survwmg the manufacturing process. This method can be rehable xf low temperatures are used d u n n g the fermentation and if good m a n u f a c t u n n g practices ( G M P ) have been conscientiously followed during preparauon of the inoculating material. This procedure may also serve as an inadvertent source of undesirable organisms in the unnpened product. Back inoculation cannot be recommended for the additional reason that desirable orgamsms present in sausages following an extended drying period may not necessarily be very well-adapted to carrying out the dependable fermentation of freshly salted meat. This technique is rarely used today in modern meat plants in Canada. The third method ~s the use of commercmlly avadable starter cultures winch compete with and actively inhibit the development of undesirable bacteria dunng fermentauon of meat (Nlskanen and Nurrm, 1976; Llepe, 1983; Bacus and Brown, 1985a,b, Lucke, 1986). Although starter culture use has

58 gamed in popularity in North America and Northern Europe, their adoption for use in the manufacture of low acid, delicately flavoured Italian dry sausages has been slow. This is undoubtedly due to the lack of statable cultures which will yield acceptable acidulation of the meat and at the same t~me allow proper color and flavor development (Cantom and Bersanl, 1985) during these low temperature ( < 2 0 ° C ) extended fermentations. Gokalp and Ockerman (1985) found that a starter consisting of L. plantarum and Mtcrococcus spp. was unsuitable for fermentations under 2 0 ° C . Lucke and Heckelman (1986) advised against the use of starters m sausages manufactured in this manner. In contrast, Raccah (1981) claimed that Lactacel 75 (P pentosaceus) was effective in Genoa sausage at 21°C in reducing vlablhty of S aureus inoculated at 104/g, but better results were obtained at higher (27 o C) temperatures. Pedlococci would not be considered good candidates as starters for tradxt~onally acceptable Italian dry sausage because of the emphasis placed on low acidity (Cantonl and Bersam, 1985). In the absence of suitable starter cultures, the manufacture of traditional Genoa and salamettl sausages in North America is evolving toward processes where higher fermentation temperatures are used with the currently available pool of starter bacteria (Tables I and II). The question, of course, arises as to whether this new generation of starter-fermented products are safer than those manufactured by the tradmonal process. Further comment will be directed toward answering this question. One of the other major &fferences between the starter and traditional processes studied was the use of a slightly higher concentration of NaC1 in the latter (3.1 versus 3.3%, respectively). These concentrations of NaC1 in themselves are not inhibitory to either staphylococci or salmonellae, but tngher salt concentrations tend to accelerate reducuons in a,, which slgmflcantly reduce the ability of both these organisms to grow during ripening. The higher NaC1 concentratxon, on the other hand, does have an m h l b i t o ~ effect upon lactic acid bacteria. Two groups, Petaja et al., (1985) and Marcy et al (1985) have shown that concentrations of about 2.5% NaCI allowed faster reductions in pH in ripening dry sausages than occurred at higher levels of NaCI. This had the redirect effect of causing a more efficient inhibition of inoculated staphylococci at the lower salt concentration but yielded a shghtly higher ultimate a,, Chllders et al, (1982) also found that a,, was slightly elevated when the salt concentration was reduced, Tile a,, of Genoa and salamettl prepared with either 3.1 or 3.3% NaC1 (Table III) were not sigmflcantly different. Finished sausage a,, values were all within the German guldehnes (Lelstner, 1978) for shelf-stable products which are also recogmzed by the USDA (Bacus and Brown, 1985a) but according to the Canadian guidelines (Agriculture Canada, 1983) shelfstable products should have a pH of < 5.4 and an a,, of 0.90 or less before release. Three samples of Genoa sausage made without starter cultures (plants G, H and I) had mean a,, values of 0.923, 0.931 and 0.944, respectively. Two samples of salametti (average of 5 analyses each) had a,~ values of 0.921 and 0.914 at plants E and H, respectively. Individual pH values of these high a,~ sausages ranged from 4.53 to 4.96. Mean numbers of staphylococci in sausages during ripening and in finished products were substantially lower (Table III) than the log10 6 - 7 which are known to

59 be hazardous to human health (Barber and Delbel, 1972; N~skanen and Nurml, 1976; Metaxopoulos et al., 1981). Individual analys~s showed that numbers of staphylococci in the raw meat used m sausage preparation were < 1000/g which IS considered normal (Metaxopoulos et al., 1981). M a x i m u m numbers of staphylococci consistently occurred in sausage meat following fermentation regardless of sausage type and method of manufacture. In Genoa sausage these numbers were sigmficantly higher ( P < 0.05) in starter-acidulated than in traditional sausages (Table III). At plants A and B the numbers of staphylococci in one experimental series reached log10 4.24 and 5.12/g at the end of fermentation of salametti, but numbers in all finished products were substantially lower. Numbers of staphylococci m fermented Genoa at plant B were also high (lOgl0 3.99) but were well below hazardous levels All samples of finished sausage, except for one lot of salamettl at plant B with log m 4.57, contained < log~0 3.0 staphylococci. It is important to note that dunng this work individual lots of sausage were not followed throughout the production process and therefore samples of finished sausage may not have been from identical lots of fermented sausages. Nevertheless, large numbers of randomly selected samples were chosen and results obtained should reflect the overall microbial content of each sampled category of product. Slightly higher numbers of staphylococci in several samples of salametti sausages prepared with starter cultures at elevated temperature (than in similarly prepared G e n o a samples of larger diameter) reflects earlier observations that staphylococcal growth is more substantial in cured sausage meat under aerobic conditions (Lee et al, 1977, Labots, 1977; Raccach, 1986). Notermans and Heuvelman (1983) found that neither growth nor enterotoxln production by staphylococci occurred at 1 8 ° C but did occur at 2 4 ° C and a,, 0.87. It was notable that in traditionally prepared sausages fermented at 2 7 - 3 4 ° C at plant G, staphylococci were not detectable In any analyzed samples. That is not to imply that this type of process is particularly safe but rather underlines the importance of care in the selection of top quality raw materials for uncooked dry sausage production. Enterotoxin analyses showed a predominance of staphylococcal enterotoxin A (SEA) among positive isolates. It seems that production of enterotoxan B (SEB) is more sensitive to NaC1 concentration (Nlskanen and Nurmi, 1976, Troller, 1986) It should also be borne in mind that surface sampling was not conducted for staphylococci although partial cross-sections of sausages were used. This technique might dilute to some extent the actual numbers of enterotoxtgenic staphylococci present in samples. Staphylococci are known to be more toxigenic during aerobic growth or within 0.3 cm from the sausage surface (Lee et al., 1977; Bacus, 1984; Sohs et al., 1984; Raccach, 1986). Sausage meat was not analyzed during this present study for enterotoxln content since numbers of potentially toxigemc staphylococci were so low in tested products (Table III). It is evident that the more slowly fermentations are conducted the less inhibitory is the antagonistic action of lactic bacteria toward staphylococci and salmonellae (Daly et al,, 1973; Smith et al., 1983). This IS probably the result of reduced production of lactic acid by lactic acid bacteria (LAB) at the lower temperatures involved in slow fermentations. Other factors are important in the prevention of

60 growth by these pathogens (Le~stner, 1985; L~epe, 1983). Salt can interact w~th m t n t e (150-200 ppm) to rather effectively reduce the levels of Salmonella in meat and n p e m n g dry sausage (Sirvlo et al., 1977; Szczawmslo et al., 1985) at 2 0 - 2 2 ° C although at high contamlnat~on levels ( > 10S/g) they may not be elinunated completely. S~rvio et al., (1977) found that w~thin 32 days, 104 salmonellae/g meat were ehminated when either Duploferment (lactobacllh and micrococc0 or Baktoferment (nucrococc0 starters were used at 10 v cells/g or when m t n t e at 150 p p m was used alone m dry sausage. Salmonella grew to levels of 105/g where no starters were used or when nitrate (300 ppm) was used by itself. The latter workers used a procedure where sausages were kept for 3 days at 1 8 - 2 0 ° C followed by 4 days at 2 0 - 2 2 ° C with drying at 1 5 - 1 7 ° C for up to 3 weeks. Thus, It appears that use of high quahty raw meats ~s more ~mportant to product safety than the use of starters during low temperature fermentations as suggested by Lucke and Hechelman (1986). Certainly, other work (Gokalp and Ockerman, 1985) as well as our own (Holley, unpubhshed) have indicated that temperatures > 20 ° C should be used for Lactobaclllus plantarum-contalning starters because presently available Lactobactllus starters do not acidulate very well below 2 0 ° C , A further study of streptococci ~solated from sausage samples and an apprmsal of the suitability of the starters used for Itahan dry sausage manufacture are presented m a companion paper. (Holley et a l , 1988b).

Conclusion It ~s hkely that the new generation of starter-fermented Itahan dry sausages manufactured at elevated temperatures are no safer rmcrobxologlcally than those manufactured by traditional techmques using a low temperature fermentauon. Neither process is sufficiently restrictive to ehminate high levels of naturally occurring staphylococc~ or salmonellae. It ~s therefore ~mportant to recogmze that during Itahan dry sausage manufacture only raw materials of the highest possible quahty be used. Regulatory authormes should also be aware that as a result of nucrobml proteolysis in low temperature ripened sausages, the p H can eventually rise and reach levels similar to those of unripened meat (Bianchi et al, 1985; Cantom and Bersani, 1985) before sausages are released for sale. These products are still safe for consumption because of low aw (Lelstner, 1985).

Acknowledgements The authors wish to acknowledge the cooperation of management at each of the plants revolved m the study, Agriculture Canada Inspection staff m both Montreal and Toronto for taking samples according to the planned protocol under the supervision of Dr. J. Trolaen and Dr. A. Maulhm, respectively. The competent analytical work of staff at Blo-lalonde Food Laboratories, Cardinal Laboratories, the Animal Pathology Laboratory and the Food Research Centre xs greatly apprecl-

61

ated. Funds were provided by the Food Production and Inspection Branch, Agriculture Canada, under contract for most of the laboratory analyses. Food Research Centre contribution number 737

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