Lactic acid bacteria in meat fermentation

Lactic acid bacteria in meat fermentation

FEMS MicrobiolosyReviews87 (1990) 165-174 Publishedby Elsevier 165 FEMSRE 00167 Lactic acid bacteria in meat fermentation Walter P. H a m m e s 1, ...

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FEMS MicrobiolosyReviews87 (1990) 165-174 Publishedby Elsevier

165

FEMSRE 00167

Lactic acid bacteria in meat fermentation Walter P. H a m m e s 1, Annegret Banfleon 2 and Seunghwa M i n 1 I Universitltt Hohenheira, l n s t i t u t ~ r Lcbensmitt¢ltechnologie StuttS~-t , F.~.G., and 2 HFds AG, Marl, F.R.G.

Key words: Fermented sausages; Starter cui~ures; Physiological properties, useful; Lactobacillus cwvatus; Lactobacillus sake

1. SUMMARY

2. INTRODUCTION

The main fermented meat products are fermented sausages in which lactic acid bacteria (LAB) are the essential agents of the ripening process. During indigenous fermentations Lactobacillus cuwatus and L sake are the dominating LAB. Their application as starter organisms ensures the dominance of the starter during the whole ripening process. The suppression of the competing fortuitous LAB depends on the quality of the raw materials and on technological factors. The physiological properties of lactic starters do not suffice to ensure a sensory quality which can be found in traditionally produced dry fermented sausages. Additional activities required are present in miorococci and yeasts which, therefore, are further components of starter ~aiture preparations. Some strains of meat-borne lactobaciili exhibit the essential activities like nitrate reductas¢, nitrite reductase, oatalase, lipase, and protease, respeotively. To create the optimal starter cultures composed of lactobacilli, these activities have to be studied and optimized in strains of high competitiveness in the fermenting substrate.

Meat is an important part of our diet and its storage requires special measures since it is highly sensitive to microbial spoilage. Factors contributing to this property are: high water activity (a w ffi 0.96-0.97); a favourable pH (5.6-5.8); and the availability of virtually all nutrients, growth factors and minerals required for optimum microbial growth. Traditional methods for preservation of meat are drying, salting and fermentation. The latter process can be traced back to Babylonian times and was practised in Asia and Europe. The main fermented products consumed nowadays in the western world are fermented sausages which have their origin in the mediterranean world from where they spread and developed into a vast variety of types. For example, 330 different types are produced in Germany [1]. To all products the following definition can be applied: fermented sausages are cured meat products that are shelf stable (without cooling) and are commonly consumed without application of any heating process. They are spreadable or become sliceable during a ripening process which involves fermentation and reduction of the waterc0ntent by drying. 3. THE ROLE OF LACTIC ACID BACTERIA IN MEAT FERMENTATION

Correspondenceto: W.P. Hanum~ Instltut flit-Leba~mitte|t~mologie, Univer~tat H o h ~ z ~ Oarlmmr. 25, D-7000 Stuttgart 70, F.R.O.

Factors affecting the quality of fermented sausages are shown in Fig. 1. Each of these factors

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166

exogenous factors

raw materials - meat type(pork, bw/. mutton) -meat quality{eg,aw, pH, collagen content) - fat content - Imrticle size casing material diameter curing agents -Salt *nitrate -nitrite spices additives

climate -temperature -relative humidity -movement of air smoke

eg.ascorbic acid sodium glutamate glucono.b-lactone sugor *r culture

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Fig. 1. Factorsaffectingthe ripeningprocessand the qualityof fermentedsausages. has an influence on the fermentative transformation of the raw materials into the desired end product durin 8 the ripening process. The aims of this process are the following: reddenin5 and acquisition of flavour, sliceability and resistance to spoilage. The technology of sansage production and the microbiology of the fermentation process have been reviewed recently [2-5]. It is remarkable that in the traditional fermentation process bacteria, yeasts and fungi contribute together and specifically to the quality of the fermented sausages, although lactic acid bacteria (LAB) would contribute to all aims of the ripening process only by themselves. In modern technology, LAB can indeed control this process as sole agent of fermentation. Their essential role was recogniT~! first in the U.S.A. whore F.::ents for the application of Lactobacillus plantarum, L. brevis and L. fermenti, were obtained by Jensen and Paddock in 1940 [6]. The special interest in LAB is related to the type of fermented sausage that is preferentially produced in the U.S.A. and that is a semi-dry pf~Jcluct (e.g. summer sausage) made with the aid of nitrite cure. It requires a

quick (12-24 h) decrease h~ pH to 4.8-5.0 and may be heated afterwards up to 74°C, thus stopping further microbial activities. The first starter culture commercially available was introduced in 1957 [7] and contah~cd Pediococcus cerevisiae, later on classified as P. acidilactici [8]. On the other hand, in Europe dry sausages with a usually milder taste are traditionally the main products, which are made with a nitrate cure and fermented for weeks or even months. For example, Schmidhofer [9] investigated 680 dry Italian type sausages and observed pH values ranging from 4.6 to 6.8 and exhibiting an average pH of 5.5. Therefore, less attention was paid to LAB and instead m i c r 0 c o ~ were first proposed as starter cultures [10]. Thereafter, in addition to pedi0cocci, L plantarum was introduced as starter organism in Europe (1966) (for review see [11]) as well as in the U.S.A. [12]. The former selection of pediococci as the preferred lactic acid starter organisms in the U.S.A. was not based on their important role in the indigenous fermentation process but rather on their good survival during preparation of freezedried cultures. A thoroush investigation of LAB in both the commercial products and in ripening sausages was performed by Reuter [13]. The dominating LAB were atypical streptobacterieb which were psychrophilic and less acid tolerant (pH limit 3.9-4.1) than typical streptobacteria (.oH limit 3.7-3.8). They outnumbered the typical lactobacilli by a factor of nearly 10 3. The latter group was composed of L. plantarum, I.~ brevis, L, alimentarius, L. casei, L, farciminis and L, viridescens together with unspecified leuconostocs and pediococci [14]. According to Kagermcier [15], the majority of the atypical streptobacteria can be allotted to L sake and L. curvatus. Reuter [16] produced fermented sausages with starters containing either typical or atypical streptobacteria and observed that some strains of the latter group led to products with excellent sensory qualifies. Thereafter, strains of I_. sake and L curvatus have been incorporated into commercial starter culture preparations. We have analysed 37 preparations available from 12 suppliers to the German market [17] and identified the organisms shown in Table 1 as components of single.strain or multiple-strain cul-

167 Table 1 Components of starter cultures for meat fermentations [2] Group Lactic acid bacteria

Mdcrocoecaceae

Streptomycetes Ye~s-'.

Fungi

(i) the initial LAB content of the raw material. The higher this LAB content, the lower the dif,ference between their concentration and that of the starter organisms in the course of the fermentation [19],

Microorganisms Pediococcus acidilactici Pediococcuspentosaceus Lactobacillus plantarum Lactobacillus sake Lactobacillus curvatus Micrococcus varians Staphylococcus carnosus Staphylococcus xylosu~ Streptomyees griseus Debaromyces hansenii Candida famata Penicillium nalgio~nse Penicillium crysogenurn

(ii) the size of the s~rter inoculum. From Fig. 2 it can be seen that 10 ~ to 10 7 cfu/g have to be applied to obtain a safe dominance of the starter organism, (iii) the formula. As shown in Fig. 3, with an increase in the concentration of dextrose added, the growth of the fortuitous LAB is improved and, concomitantly, the final pH decreased; (iiii) the types of the competing LAB. In Fig. 4 it is shown that the connnonly well competing strain L. curvatus Ix 2 performedquite differently when

tures. The high number of available starter preparations does not imply that a corresponding multitude of starter strains are in use. We have studied the plasmid profiles of the isolates from these starter preparations and have found that for each group of isolates, which could be allotted to L plantarum, P. pentosaceus or P. acidilactici, only two plasmid profiles were detectable (W.P. Hammes, unpubfished results).

4. L A C T O B A C I L L U S CURVATUS S A K E AS S T A R T E R O R G A N I S M S

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L.

Based on the observation of Router that L. curvatus a n d L. s a k e are t h e most competitive

LAB in sausage fermentations and that it is possible to produce good quality sausages with these organisms, we investigated the effect of these organisms on the microbiology of fermenting sausages. It was observed that strains of these organisms isolated from fermented sausages usually grew well in the sausage mixture and dominated the fortuitous population during the whole ripening process [18]. Strains isolated from non-meat environments (e.g. the type strains of L. s a k e a n d L curvatus) did not exhibit this competitiveness. The ability of strains of L. curvatus or L. s a k e to dominate the fortuitous LAB depended on the following factors:

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symbols represent the growth of the corresponding fortuitous LAB.

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challenged with inocula of either another L. curvatus, L sake or a starter derived strain of P. acidilactici. For example, in the presence of strain L. curvatus Lc 5 strain Lc 2 lost the competition. In the presence of L sake Ls 10 or P. acidilactici, however, Lc 2 h a d a competitive advantage. It is thus clear that the performance of the starter LAB m a y b e affected if a strongly competitive population of LAB is present in the raw material. Therefore, to prevent the possible risk of producing poor quality sausages even in the presence of starters, it is still necessary to employ raw materials of low initial bacterial load a n d to be sure that sufficient starter culture is added.

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5. D E S I R E D P H Y S I O L O G I C A L P R O P E R T I E S O F LAB I N S T A R T E R C U L T U R E S

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t i m e of r i p e n i n g (d) F i ~ 3. The effect o f ~ue4)~ c,,oncenfxatioll ou the growth o | ].~ curvatgs Lc 2 used as staz1~r (solid symbols) and the fortuitous LAB (A), and (B) on the decrease of pH. The symbols in A

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LAB starter cultures affect the quality of the fermented sausages in several ways. Of major importance is the formation of lactic acid from the



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t i m e of ripening (d) Fig. 4. Competition between LAB in fermentin8 sansases. In the ¢~xperimentsdepicted in A-D L. curvatus Lc 2 was used as inoculum (AI, A). A. The growth of the fortuitous LAB in the absence (o o) or presence ( O ~ O ) of strain Lc 2. B. The 8rowth of/.. curva~ Lc 5 in the absence (@ o) or presence (11 II) of strain Lc 2 ( A ~ A ) . The open symbols represent the conesponding growth of the fortuitous LAB in the two sets of experiments. C. The growth of L sake Ls 10 in the absence (o e) or presence (11 am)of strain Ix 2 ( A ~ A ) ; open symbols, see B. D. The growth of P. acidilacticiin the absence (e~e) or prescnce (m ii) of strain Lc 2 (A A); open symbols, see B,

169

added carbohydrate source. The resulting decrease in pH causes the following effects: (i) coagulation of the meat proteins whereby the texture changes and the sausages become sficeable; (ii) acid taste; (iii) improved hygienic stability; ('fiii) reddening as a result of an acid catalysed disproportioning of nitrite according to the equation 3 HNO ~- 2 N O + H N O + H20 Nitrogen monoxide reacts subsequently with myoglobin to finally form the cured meat colour nitrosomyoglobin. For long fermenting dry sausages it is commonly accepted that LAB as the sole starter organisms do not yield the aromatic and sensory appealing products which can be obtained when micrococci and yeast are applied additionally. Activities that are regarded as properties of the nonlactic starter organisms are: nitrate reductase, nitrite reductase, catalase and lipase. However, these activities can also be found in LAB strains involved in meat fermentation, as shown in Table 2. For both catalase and nitrite reductase, activities were found that are either heme-dependent or -independent. Since meat contains sufficient heme, there should be no limitation for heine-dependent activities in fermenting sausages. It is remarkable that nitrite is reduced to ammonia by the hemedependent nitrite reductase whereas the heine-independent enzyme releases N20 and NO as prod-

ucts. The latter activity can, therefore, contribute to an improved reddening. We have studied whether the nitrate and nitrite reductase activities of lactobacilli can be sufficiently hish to ensure the required low content of nitrate in the final product. Sausages containing either nitrate or nitrite were inoculated with L. p e n t o s u s , exhibiting nitrate reductase activity, and with strains of I . s a k e a n d L f a r c i m i n i s , p e r forming nitrite reduction. As shown in Fig. 5, the multiple strain culture achieved the reduction of nitrate and nitrite (expressed as concentration of total nitrate) even in the sausage prepared with nitrate. However, the drop was only slow and, therefore, colour and flavour defects resulted. With L s a k e I.s 8 alone, the reduction was not sufficient. It is characteristic for all strains of L curvatus that they are devoid of catalase activity whereas this activity is present in L s a k e [21]. This result is consistent with the observation that with the latter bacterium as starter a greyish surface discolouration has not been observed. This defect may, however, occur with L c u r v a t u s a s s t a r t e r a n d should result from the formation of H202 near the surface where oxygen has access to the fermenting substrate. Lipolysis is generally considered to play a key role in the development of aroma and has been studied in fermenting sausages by Cantoni et al. [27]. These authors attribute lipolysis in sausages to the properties of micrococci. On the other hand, Renter [28] has shown that strains of L. s a k e a n d

Table 2 Rare propertiesof meat-bornelacticacid bacteriawith favourableeffects on the fermentationof raw sausages Reference LAB Catalase NitrateNitritereductase reductase Heine Heme Heme Heine dependent independent dependent independent 1. plantarurn + (+) (+) (+) (+) [20,23,24,21,261 L. pentosus + -+ + [21] L farciminis L sake

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nitrate or nitrite as curing aids. The cot~'~ttation of nitrite and nitrate were determined and expressed as total nitrate. Multiplestrain starter cultureswere used (10vcfu/g) containin8/.. pentosus,DSM 20314(nitratereductas¢ +, catalase +), L farciminis, 1136 (nitrite reductas¢ +) and L. sake Ls 8 (nitrite reduetase +, catalese +). in sausagesprepared with nitrate ([3 [3) or nitrite (0 0). A single strain culturecon~iv, g of L sake, Ls 8 was used (0 o) as a control. 1~ curvatus exhibit lipolytic activity in vitro and

further that pediococci and the typical strcptobacteria are devoid of that property, The importance of lipolysis by the lactobacilli, however, has not yet been studied in fermenting sausages. Even less is known about the role of proteolysis during the fermentation, although in vitro studies have shown that proteolytic activity can be found in strains of both typical and atypical streptobacteria [291. Meat-borne strains of Lactobacillus have been investigated for their potential to produce antagonistic compounds. This property has indeed been detected in strains of L. curvatus, L plantarum, and I.. sake. A compound, named

sakacln A, was produced by a strain of L. sake. It was found to be of proteinaceous nature and its synthesis appeared plasmid-encoded [30]. As shown in Table 3, we have observed that strains of L curvatus and L sake inhibit not only food-poisoning bacteria but also strains which are commonly incorporated into multiple-strain starter preparations. Thus, the knowledge of these properties provides a means of selecting the optimum combinations of starter strains that reduce potential hygienic risks in fermenting meat products. This knowledge is also important to avoid a combination of those strains of L sake which exhibited a strong antagonism against M. varians. In fact, we have observed that nitrate reduction and the reddening reaction were suppressed in sausages, when such a combination was used. We have further studied whether strains of L sake exhibiting activity against $. aureus can suppress the growth of this food-poisoning organism in ripening sausages. As shown in Fig. 6, the application of the L sake strains reduced strongly the numbers of $. aureus in both spreadable and sliceable sausages. Corresponding practical studies with Listeria monocytogenes did not reveal a special inhibiting effect of strains of L sake which exhibited an in vitro activity against this food pathogen. The growth of S. aureus in fermented sausages may be a risk when certain types of sausages are produced. A quick decrease of pH to values below 5.0 is a means of safely reducing this risk. In traditionally produced fermented dry sausages problems arising from the growth of S. aureus have not been reported. It was shown [32] that the combined application of lactobacilli, micrococci and yeasts acts synergistically in suppressing the growth of 8. aureus in fermented sausages. It was suggested that each of the microbial groups contributes characteristically to this effect by decreasing the pH, producing nitrite and decreasing the rcdox potential, respectively. Antagonistic compounds may also be involved in enhancing the favourable effect of the starter culture. Different from starters in the dairy field, the resistance to phase attack has not been considered an important property of lactic starters for meat fermentation. The solid matrix appears to prevent

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the spread of phages during the fermentation process. When lactic starters were applied in sausage production togather with their phage, only a short delay in ripening was detected [33]. In addition, starters are usually not propagated in places where the meat is fermented and therefore the risk of phage infections of the cultures is low.

6. CONCLUDING REMARKS The study of the effect of LAB starter cultures has revealed that their application ganerally ensures that the aims of the ripening process are

attained more safely than it was possible in the traditional spontaneous fermentations. On the other hand, to obtain dry fermented sausages of high sensorial quality, multiple-strain preparations containing non-lactic cultures as well are still required. It is not yet sufficiently known which physiological properties of the lactic starters can be usefully employed to simplify the microbiology of the fermentation process and to obtain the optimum quality of sausages. Besides a high competitiveness of the starter strains and a safe decrease in pH, activities that improve flavour, colour, and hygienic safety should be targets of future search for new strains of LAB. Apparently strains of L sake and L curvatus are good candidates for further research. These bacteria may either be combined in multiple-strain lactic starters that contain the various activities or may receive enhanced and even new enzyme activities with the aid of genetic modification. However, the genetics of the meat-specific lactobacilli have been investigated only poorly. It has been shown that the methods developed for genetic modification of lactococci or L casei can be adapted to L curvatus and L sake [34]. Since virtually all of those physiological properties are present in the various species of lactobacilli that are discussed as essential prerequisites for achieving optimum quality of fermented sausages, it appears even feasible to employ this potential for intrageneric gene transfer. The resulting LAB should combine the high competitiveness with an enlarged physiological activity. This activity may be modified in the various starter strains to take in consideration the special requirements for the production of the various types of fermented sausages.

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