Biochemical and sensory characteristics of traditional fermented sausages of Vallo di Diano (Southern Italy) as affected by the use of starter cultures

In this study, two strains of Staphylococcus xylosus isolated from traditional fermented sausages of Vallo di Diano (Southern Italy) were used in combination with an acidifying strain of Lactobacillus curvatus as starter culture for the production of fermented sausages. Two starter formulation were developed combining the proteolytic but not lipolytic (prt(+), lip(-)) S. xylosus CVS11 with the L. curvatus AVL3 (starter S1) and the S. xylosus FVS21 (prt(-), lip(+)) with the same strain of L. curvatus (starter S2). Proteolysis and lipolysis were observed during ripening by the increase in total free amino acids (FAA) and free fatty acids (FFA), respectively. Such activities were observed in both started and non started sausages (control). Moreover, the proteolytic and lipolytic activities were detected in products started by both formulations irrespective of the presence of such activities in the strains used. Therefore, it was not possible to conclude whether the effect of proteolysis and lipolysis during ripening of the started fermented sausages was due to the activity of the starter cultures or to the action of meat endogenous enzymes.     

Use of Fourier transform infrared spectroscopy to evaluate the proteolytic activity of Yarrowia lipolytica and its contribution to cheese ripening

The interaction between tyrosine-decarboxylase and proteolytic activities of a Lactobacillus curvatus and Staphylococcus xylosus, respectively, on biogenic amine production during the ripening and the storage of dry fermented sausages was investigated. Water content, pH, proteolysis parameters, microbial counts, and biogenic amine contents were monitored in spontaneously and starter fermented sausages. The use of proteolytic staphylococci as starter resulted in a higher content of non-protein nitrogen and total free amino acids. Tyramine was the main amine produced in all batches. However, tyrosine-decarboxylase activity of the L. curvatus starter strain was weak and yielded lower amounts of tyramine than those produced by the wild mioroflora in the control batch. Association between tyramine production and proteolysis could only be established in a defectively dried batch. Putrescine and cadaverine accumulation was efficiently reduced in the starter-mediated fermentation, in agreement with the lower development of enterobacteria. Phenylethylamine and tryptamine were only detected in the spontaneously fermented sausages, while histamine, spermine and spermidine did not vary during the ripening. Biogenic amine levels and related parameters showed significant changes during the storage of dry sausages depending on the temperature and the batch. As a general rule, changes in the pH, proteolysis, microbial counts, and biogenic amine contents were stronger at 19 degrees C than at 4 degrees C. The results suggest that refrigeration would be advisable for preventing further accumulation of biogenic amines during the storage of dry fermented sausages.     

Role of surface-inoculated Debaryomyces hansenii and Yarrowia lipolytica strains in dried fermented sausage manufacture. Part 1: Evaluation of their effects on microbial evolution, lipolytic and proteolytic patterns

The aim of this work was to study the effects of Debaryomyces hansenii and Yarrowia lipolytica strains, used with lactic acid starter cultures (Lactobacillus plantarum), on the manufacture of dried fermented sausages to understand their role on sausage microbial evolution, lipolytic and proteolytic patterns. The inoculation of the yeast strains did not markedly affect the sausage’s microbial flora. The sausages with the yeast strains showed more marked and earlier water activity (a_w) reductions. Moreover, the surface inoculation of the yeast strains resulted, at the end of ripening, in more pronounced proteolysis and lipolysis. The lipolytic patterns of the products were affected not only by the yeast strain but also by the level of mincing of the meat mixture used.     

Aroma components from dried sausages fermented with Staphylococcus xylosus

Sausages with and without Staphylococcus xylosus were manufactured with four replicates. Antibiotics and a fungicide to inhibit growth of naturally occuring microorganisms were added to the control sausages. The volatile compounds from the sausages were collected and identified by gas chromatography/mass spectrometry, the effluent evaluated by sniffing and the sausages assessed by a trained panel. The study showed that sausages fermented with Staphylococcus xylosus contained several fragrant esters that were not found in control sausages without microbial growth. Control sausages had an unpleasant, rancid odour compared to sausages with Staphylococcus xylosus, indicating that the esterase activity of Staphylococcus xylosus or other microorganisms is very important in order to obtain the proper fermented sausage aroma. Although sausages with Staphylococcus xylosus contained the highest amounts of free fatty acids, it seemed to be of no importance to the aroma development. It is therefore questionable whether lipolytic activity of starter cultures has any influence on sausage flavour.     

Lipolytic Starter Culture Effects on Production of Free Fatty Acids in Fermented Sausages

Fermented sausages, inoculated with lipolytic and non lipolytic starter cultures and a control containing glucono-δ-lactone (GDL) and an antibiotic mixture were prepared. The sausages were analyzed on the basis of color, pH, water activity, weight loss, moisture and fat contents. Microbiological analyses were performed to measure total viable counts, lactobacilli and, staphylococci and micrococci. The increase in total and individual free fatty acids, and lipid oxidation was monitored. Results showed no difference (p>0.05) in the level of free fatty acids between treatments, indicating that lipolysis was most likely due to endogenous meat enzymes.     

Changes in selected biochemical and sensory parameters as affected by the superficial inoculation of Penicillium camemberti on dry fermented sausages

An atoxigenic strain of Penicillium camemberti was superficially inoculated on fermented sausages in an attempt to improve their sensory properties. The growth of this mould on the surface of the sausages resulted in an intense proteolysis and lipolysis, which caused an increase in the concentration of free amino acids, free fatty acids (FFA) and volatile compounds. Many of these were derived from amino acid catabolism and were responsible for the "ripened flavour", i.e. branched aldehydes and the corresponding alcohols, acids and esters. The development of the fungal mycelia on the surface of the sausages also protected lipids from oxidation, resulting in both lower 2-thiobarbituric acid (TBARS) values and lipid oxidation-derived compounds, such as aliphatic aldehydes and alcohols. The sensory analysis of superficially inoculated sausages showed clear improvements in odour and flavour and, as a consequence, in the overall quality of the sausages. Therefore, this strain is proposed as a potential starter culture for dry fermented sausage production.     

Characterization and selection of autochthonous lactic acid bacteria isolated from traditional Iberian dry-fermented salchichón and chorizo sausages

ABSTRACT:  A total of 192 lactic acid bacteria were isolated from 2 types of naturally fermented dry sausages at 3 different stages of the ripening process in order to select the most suitable strains as starter cultures in dry-cured sausage manufacture according to their technological characteristics such as glucose fermentation, lactic and acetic acid production, and proteolytic, lipolytic, and antimicrobial activities. Identification of the isolates revealed that 31.2% were Pediococcus pentosaceus , 26.9% Lactococcus lactis , 18.6% Pediococcus acidilactici , 17% Lactobacillus brevis , and sporadic isolates of Leuconostoc mesenteroides , Lactobacillus plantarum , and Lactobacillus curvatus . Most of the strains did not produce gas from glucose and showed the capacity to produce lactic acid rapidly. Some 25% of the strains were able to degrade tributyrin (esterase activity), but none showed lipolytic activity against olive oil and pork fat. Only 3 strains of P. acidilactici showed weak proteolytic activity against myofibrillar or sarcoplasmic proteins. Also, the same strains showed antimicrobial activity against Listeria monocytogenes . Nine strains with the best properties were preselected and tested for biogenic amine production. The results showed that two of the strains, identified as P. acidilactici by polymerase chain reaction, had the potential to be further tested as starter cultures in pilot processing of Iberian sausages.     

Isolation and technological properties of coagulase negative staphylococci from fermented sausages of Southern Italy

The aims of this study were to characterize the population of Micrococcaceae in different types of fermented sausages of Southern Italy and to determine the technological properties of Staphylococcus strains in order to evaluate the suitability of selected strains as starter cultures in the processing of dry fermented pork sausages. Ninety-six strains were studied to evaluate nitrate reductase, proteolytic, lipolytic and antioxidant activities as well as growth ability at different temperatures, pH's and NaCl concentrations. All the strains were classified as Staphylococcus except for one isolate assigned to Kocuria spp. The species most often isolated were S. saprophyticus, S. xylosus and S. equorum, although they were not equally distributed within the different sausages. Other species isolated were, in descending order of abundance, S. succinus, S. warneri, S. lentus, S. vitulus, S. pasteuri, S. epidermidis, and S. haemolyticus. In general, the S. xylosus strains exhibited the best technological properties that would make them eligible as good starter cultures for fermented meat products. However, strains belonging to other species also showed good technological properties. Finally, all strains grew at 10, 15 and 20 °C, in the presence of 10% and 15% of NaCl and at pH 5.0 and 5.5. The results showed that it is possible to formulate a broad variety of staphylococcal starter cultures, adaptable to different technological conditions and sausage manufacture practices.     

微生物酶与肉组织酶对干发酵香肠中游离脂肪酸的影响

以戊糖乳杆菌31-1和木糖葡萄球菌为发酵剂生产干发酵香肠,就发酵剂对干发酵香肠成熟过程中游离脂肪酸的影响进行了研究。研究结果表明,在灌肠后的1周内,脂肪的水解主要是由组织酶引起的,微生物对脂肪微弱的水解能力主要表现在干发酵香肠成熟的后期,并且发酵剂不能改变发酵香肠中游离脂肪酸的变化模式。     

Preliminary analysis of the lipase gene (gehM) expression of Staphylococcus xylosus in vitro and during fermentation of naturally fermented sausages (in situ

Coagulase-negative catalase-positive cocci (CNCPC) play a very important role during the fermentation of sausages. In particular, they are involved in the aroma formation of the final product, because they release lipases that are able to free short-chain fatty acids that are contributing to the sensory characteristics of the fermented sausage. Few studies have been undertaken to elucidate the regulation of lipase gene expression in Staphylococcus xylosus by substrate molecules or products of lipolysis. The aim of this study was to analyze the gehM gene expression of S. xylosus DSMZ 6179 in vitro with growth media containing different concentrations of lipids and in situ during the maturation of fermented sausages. The results obtained suggest that a concentration that increases in triglycerides in the growth medium suppresses the expression of the lipase gene.     

Starter strain related effects on the biochemical and sensory properties of Cheddar cheese

A detailed investigation was undertaken to determine the effects of four single starter strains, Lactococcus lactis subsp. lactis 303, Lc. lactis subsp. cremoris HP, Lc. lactis subsp. cremoris AM2, and Lactobacillus helveticus DPC4571 on the proteolytic, lipolytic and sensory characteristics of Cheddar cheese. Cheeses produced using the highly autolytic starters 4571 and AM2 positively impacted on flavour development, whereas cheeses produced from the poorly autolytic starters 303 and HP developed off-flavours. Starter selection impacted significantly on the proteolytic and sensory characteristics of the resulting Cheddar cheeses. It appeared that the autolytic and/or lipolytic properties of starter strains also influenced lipolysis, however lipolysis appeared to be limited due to a possible lack of availability or access to suitable milk fat substrates over ripening. The impact of lipolysis on the sensory characteristics of Cheddar cheese was unclear, possibly due to minimal differences in the extent of lipolysis between the cheeses at the end of ripening. As anticipated seasonal milk supply influenced both proteolysis and lipolysis in Cheddar cheese. The contribution of non-starter lactic acid bacteria towards proteolysis and lipolysis over the first 8 months of Cheddar cheese ripening was negligible.     

Meat starters have individual requirements for Mn(2+)

The effect of different Mn(2+) concentrations on sausage fermentation was evaluated. A screening experiment was carried out with six lactobacilli starters in a sausage model. To further investigate the effects found, two selected lactobacilli strains were tested in pilot-scale sausage production. For all starters an increased fermentation rate was observed after Mn(2+) addition. Differences in the development of microbial, textural and sensory parameters were observed in the sausages. For one of the cultures these differences levelled out during sausage production yielding identical end products with and without Mn(2+), for the other strain the differences due to Mn(2+) addition in the sausages remained throughout the production process yielding sausages with different properties. Knowing a starter culture's requirements for Mn(2+) will allow optimisation of dry fermented sausage production in order to increase reliability and reproducibility of production decrease fermentation time and ensure microbial safety of the final product.     

Microbial and physico-chemical changes during the ripening of dry fermented sausages superficially inoculated with or having added an intracellular cell-free extract of Penicillium aurantiogriseum

The effect of inoculation of the "salchichón" (dry fermented sausage) surface with an atoxygenic, proteolytic and lipolytic strain of Penicillium aurantiogriseum and/or the addition of an intracellular cell-free-extract (ICFE) of the same mould on the ripening process was studied. Four batches of salchichón were manufactured: control, superficially inoculated, added with the intracellular cell free extract and combination of both treatments. Superficial mould modified Micrococcaceae counts, pH, free amino acids, lipid fractions, TBARS and some organic acids. The ICFE degraded free amino acids, producing a rise in ammonia. Both treatments combined accelerate the proteolysis and lipolysis of the sausages and also the further amino acid degradation.     

Molecular and technological characterization of Staphylococcus xylosus isolated from naturally fermented Italian sausages by RAPD, Rep-PCR and Sau-PCR analysis

Coagulase-negative cocci (CNC) are important microorganisms in fermented sausages because they release lipases and proteases that are able to free short-chain fatty acids and peptides and aminoacids, respectively, that are responsible for the aroma of fermented sausage. The purpose of this study was to characterize Staphylococcus xylosus strains isolated from naturally fermented sausages, produced in three different processing plants in the Friuli Venezia Giulia region in the Northeast of Italy. Two hundred and forty-nine strains of S. xylosus were identified by species-specific PCR and subjected to molecular and technological characterization. RAPD-PCR with primer M13, Rep-PCR using primer (GTG)(5) and Sau-PCR with primer SAG(1) were used for the molecular analysis, while the capability of the strains to grow at different temperatures and in the presence of NaCl and their lipolytic and proteolytic activity were tested in order to define the technological characteristics. The results obtained allowed us to differentiate strains coming from different plants, thereby admitting the presence of strains that are plant-specific.     

Identification and characterization of potential autochthonous starter cultures from a Croatian "brand" product "Slavonski kulen"

The microbial population of a traditional Croatian fermented sausage "Slavonski kulen" was isolated, identified and subjected to technological and functional characterization in order to select potential autochthonous functional starter cultures. Dominant microflora were lactic acid bacteria (LAB), followed by staphylococci. Identification of isolated lactobacilli showed domination of Leuconostoc mesenteroides and Lactobacillus acidophilus while Staphylococcus xylosus and Staphylococcus warneri outnumbered the staphylococcal microbiota. Most of the isolated LAB and Staphylococcus species displayed good growth in the presence of 5% of NaCl and at 12, 18 and 22°C. All LAB and most of the staphylococci possess proteolytic activity and only Staphylococcus xylosus had lipolytic activity. All lactobacilli and staphylococci isolates produced significant concentrations of lactic acid (as determined by HPLC) and showed antimicrobial activity against pathogenic test microorganisms. Dominant LAB and Staphylococcus species displayed growth in the presence of 1% bile. Most of the staphylococci and all of lactobacilli showed sensitivity to all antibiotics tested.     

Dry fermented sausages elaborated with lipase from Candida cylindracea. Comparison with traditional formulations

The addition of microbial lipase to fermented sausages was studied. A sausage with lipase from Candida cylindracea and a control sausage with starter (Lactobacillus plantarum and Staphylococcus carnosus) were produced in a pilot plant. The acidity value and the amounts of the different free fatty acids (FFA) showed a higher intensity of lipolytic activity in sausages with lipase than in sausages with starter. In sausages with lipase, the percentage of saturated FFA was greater and that of polyunsaturated FFA was lower than in sausage with starter. Mono-unsaturated FFA percentage was similar in both sausages. TBA and peroxide values indicated that the increase of FFA produced by lipase action did not increase the rancidity. A slight increase in acetic, propionic and butyric acids was observed in sausage with lipase but this was not sufficient to develop excessive acidity in the product.     

Effect of prefreezing hams on endogenous enzyme activity during the processing of Iberian dry-cured hams

The use of frozen/thawed raw material in the processing of Iberian dry-cured ham has been studied to determine its effect on the sensory quality of the final product. The proteolysis and lipolysis processes were measured by the proteolytic and lipolytic enzyme activities and free amino acids and free fatty acids. The thawed Iberian hams had lower salt contents throughout the process. The use of thawing raw material did not affect the proteolytic enzymes, cathepsins, aminopeptidases and dipeptidylpeptidases, only the activity of dipeptidylpeptidase III was reduced due to thawing. Moreover, there were no differences in the content of free amino acids between fresh and thawed hams during the whole process. However, the use of thawing hams affected the lipolytic activity. The activity of phospholipase and neutral lipase were significantly higher in the thawed hams and also the content of free fatty acids, at all the stages analyzed. Consumer sensory analysis showed thawed Iberian hams had the lowest hardness, probably due to an intense proteolysis. The acceptability of the Iberian hams was similar between fresh and thawed hams.     

Influence of starter cultures on the free fatty acids during ripening in Tea sausages

The influence of starter cultures on the free fatty acids content during ripening of Tea sausages, typical dry sausages produced in the south of Croatia, was studied. Three batches of Tea sausages were produced using different starter mixtures (Staphylococcus xylosus S81 and Lactobacillus sakei G20; Staphylococcus xylosus S142 and Lactobacillus sakei G20; Staphylococcus xylosus S206 and Lactobacillus sakei G20), while the control batch was produced without a starter. The amounts of free fatty acids present in the samples at the end of the ripening period were not significantly different, suggesting that the lipolytic enzymes naturally occurring in meat could play a predominant role in the free fatty acids release. Oleic and linoleic acids were present in the highest concentrations, while only small quantities of short-chain fatty acids were detected, with acetic acid being the most representative one.     

Effects of muscle shortening and proteolysis on Warner-Bratzler shear force in beef longissimus and semitendinosus

The effect of the addition of the fungal protease EPg222 on the sensory characteristics of dry fermented sausage "salchichón" ripened with commercial starter cultures was investigated. Sausages were prepared with purified EPg222 and Staphylococcus carnosus, Staphylococcus xylosus, and Lactobacillus sakei as starter cultures, ripened for 145 days and compared with a control batch only inoculated with the starter cultures. Dry fermented sausages ripened with EPg222 and starter cultures showed higher amount of NPN and volatile compounds derived from amino acid catabolism, than control ripened only with starter cultures. Several branched aldehydes, acids and alcohols such as 2- and 3-methylbutanoic acid and 2-methylpropanol were detected only in enzyme treated samples. Sensory analysis reflected higher values for aroma intensity of sausages treated with EPg222 and lower values of hardness than control. The effect of EPg222 may be of great interest to improve sensory characteristics of dry fermented sausages ripened with starter cultures.     

微生物酶与肉组织酶对干发酵香肠中游离氨基酸的影响

以戊糖乳杆菌31-1和木糖葡萄球菌为发酵剂生产干发酵香肠,就发酵剂对干发酵香肠成熟过程中游离氨基酸的影响进行了研究,同时对干香肠在成熟过程中微生物和pH值的变化进行了分析。研究结果表明,微生物的代谢活动能增加干发酵香肠中游离氨基酸的绝对含量,但相对组织酶来说,微生物酶对游离氨基酸的解离能力要小得多,说明在干发酵香肠的成熟过程中,组织酶对蛋白质的水解起主要作用。但研究结果同时也表明,微生物对个别游离氨基酸的变化具有最大的影响。