The performance of Streptococcus macedonicus ACA-DC 198 as starter culture in Kasseri cheese production

Streptococous macedonicus ACA-DC 198 was used both as an adjunct (Cheese B) and as sole starter (Cheese C) in Kasseri cheese production. Control cheese (Cheese A) was prepared using a commercial starter culture. In all cheeses, numbers of all microbial groups examined initially increased, but declined after Baski scalding and remained practically stable up to day 90. The presence of S. macedonicus was confirmed until the end of ripening (Cheeses B and C); this was also the case for the bacteriocin produced by S. macedonicus ACA-DC 198. X-propyl-dipeptidyl-aminopeptidase activity was detected in all cheeses after day 15. The physicochemical characteristics of the mature Cheese C were in agreement with those defined by the Greek legislation. The sensory properties of all mature cheeses corresponded with the ones characterizing the traditional Kasseri cheese. It was concluded that S. macedonicus ACA-DC 198 can be used as an adjunct in Kasseri cheese production.     

Streptococcus macedonicus ACA-DC 198 produces the lantibiotic, macedocin, at temperature and pH conditions that prevail during cheese manufacture

Streptococcus macedonicus ACA-DC 198, a natural cheese isolate, produces the anticlostridial bacteriocin, macedocin. Bacteriocin activity was detected from the mid-exponential growth phase and remained constant during the stationary phase. A secondary model was setup to describe the influence of temperature (20-45 degrees C) and pH (5.1-6.9) on cell growth of and bacteriocin production by S. macedonicus ACA-DC 198 during in vitro laboratory fermentations. The optimum temperature for bacteriocin production (20-25 degrees C) was markedly lower than the optimum growth temperature (42.3 degrees C). In contrast, the specific macedocin production was maximal around pH 6.0, whereas growth was optimal at pH 6.4. Consequently, the maximum bacteriocin activity was reached between pH 6.0 and 6.5.     

Inhibition of Clostridium tyrobutyricum in cheese by Lactobacillus gasseri

A semi-hard cheese produced from milk artificially contaminated with Clostridium tyrobutyricum spores (2.5×10³ mL⁻¹) was used as a model for studying the ability of bacteriocin-producing Lactobacillus gasseri K7 (Rif^r) to inhibit clostridia. The added lactobacilli did not inhibit the primary starter culture (Streptococcus thermophilus), but inhibited non-starter mesophilic lactobacilli. Late blowing as a result of Cl. tyrobutyricum outgrowth and butyric acid fermentation occurred in all cheeses however it was reduced in cheeses with added Lb. gasseri. After 6 weeks, the average amount of butyric acid was significantly higher in cheeses without added lactobacilli (1.43 vs. 0.70 g kg⁻¹). At the end of 8-weeks ripening, 2.8×10⁷ cfu g⁻¹ of K7 (Rif^r) viable cells were detected. Using the total DNA from cheeses with added K7 (Rif^r) strain, PCR products were amplified with primers specific for Lactobacillus, Lb. gasseri and K7 bacteriocin gene.     

Streptococcus thermophilus 580 produces a bacteriocin potentially suitable for inhibition of Clostridium tyrobutyricum in hard cheese

A strain of Streptococcus thermophilus that inhibits Clostridium tyrobutyricum has been isolated from raw milk. The active compound produced disappears after a treatment with protease. However, unlike most bacteriocins, it is not thermoresistant, and the activity is completely lost after 1 h at 60 degrees C. Its inhibitory spectrum is limited to other thermophilic streptococci, Brochothrix, and sporulated gram-positive rods. So this bacteriocin could be different from those already described. This bacteriocin-producing strain could be used in thermophilic starter for hard cheese making because the bacteriocin is not active against thermophilic lactobacilli. It is produced in M17 medium during the decreasing temperature phase of the hard cheese-making process temperature cycle and is also produced in milk. Moreover, when Streptococcus thermophilus was cocultured with a Lactobacillus delbrueckii subsp. lactis starter strain, it seems to enhance the bacteriocin production. However the level of activity always decreases drastically during the stationary phase. But inhibition of Clostridium tyrobutyricum spores can be obtained in small-scale curds.     

Inhibition of Clostridium tyrobutyricum by bacteriocin-like substances produced by lactic acid bacteria

Lactic acid bacteria were selected for their inhibitory activity against Clostridium tyrobutyricum under conditions that eliminate the effects of lactic acid and hydrogen peroxide. Four strains were isolated belonging to the species Lactococcus lactis ssp. lactis. The sensitivity of the inhibitory substances to pronase and trypsine indicates that they are proteins or peptides different from nisin. Their resistance to phospholipase D indicates that they are also different from lactostrepcin. The inhibitory substances are produced during the exponential phase of growth. Their activity is bactericidal and directed toward some strains of Clostridium tyrobutyricum, Lactobacillus helveticus, and Streptococcus thermophilus, but strains used as dairy starters, Lactobacillus lactis, Streptococcus thermophilus, and Propionibacterium shermanii, are not all affected by the inhibition.     

Inhibition of Clostridium tyrobutyricum in Vidiago cheese by Lactococcus lactis ssp. lactis IPLA 729, a nisin Z producer

Lactococcus lactis ssp. lactis IPLA 729 is a nisin Z producer isolated from raw milk cheese able to grow and produce nisin Z in milk. The ability of this strain to inhibit the growth of Clostridium tyrobutyricum CECT 4011, a late blowing agent, in Vidiago cheese, a semi-hard farmhouse variety, manufactured in Asturias, Northern Spain, was investigated. For control purposes, cheeses were manufactured with the mesophilic mixed starter IPLA-001. In experimental cheeses, the nisin-producing strain L. lactis IPLA 729 was combined with this starter. Nisin Z activity reached a concentration of 1600 AU/ml in 1-day cheeses and this level was maintained until 15 days of ripening. Furthermore, to compare the inhibitory activity of the nisin-producing strain to nitrate, cheeses were also manufactured with a commercial starter culture and potassium nitrate as anti-blowing agent was added in accordance with Vidiago's cheesemakers. The control, experimental and commercial cheeses were contaminated with C. tyrobutyricum CECT 4011. The composition of the three different cheeses showed only slight differences with respect to total solids, protein and fat, although control and experimental cheeses showed a richer flavour-compound profile than commercial cheeses. The level of the spoilage strain C. tyrobutyricum CECT 4011 decreased from 1.2x10(6) to 1.3x10(3) cfu/g during ripening in presence of the nisin Z producer, while it increased to 1.99x10(9) cfu/g in control cheeses and to 3.5x10(7) cfu/g in commercial cheeses.     

Effect of production factors and ripening conditions on the characteristics of Serra cheese

Summary  The individual and interactive effects of four production factors (amount of vegetable rennet, temperature of coagulation, pressing and salting of the fresh cheese) and two ripening factors (temperature and relative humidity) on microbiological, physico-chemical, biochemical, textural and sensory characteristics of Serra cheese were simultaneously studied using a 2vi^6-1 factorial design. Highly significant effects of salting and ripening relative humidity upon the characteristics of the cheese were detected. Addition of salt to the surface of the fresh cheese reduced microbial growth, water activity, moisture and lactic acid contents, proteolysis, lipolysis, aroma and softness of the cheese. Conversely, increase of the relative humidity during ripening increased these charactereristics. Pressing had no statistically significant effect on cheese characteristics.     

Influence of milk centrifugation, brining and ripening conditions in preventing gas formation by Clostridium spp. in Gouda cheese

This study examined milk centrifugation, increased salt concentration, and low ripening temperature as potential strategies to prevent late blowing caused by gas-forming Clostridium spp. in Gouda cheese. The survival of clostridia spores in cheese brine and their ability to enter Gouda cheese during brining was also evaluated. Centrifugation (3000 x g for 30 s) of contaminated milk resulted in > 60% spore reduction, with increased spore reduction at greater centrifugal forces. Low levels of C. tyrobutyricum and C. sporogenes spores survived in saturated (23%, w/v) brine with 2% (v/v) added whey at 15 degrees C for 63 days, while C. beijerinckii and C. butyricum spores were not detectable on days 4 and 35, respectively. Spores of C. tyrobutyricum in brine infiltrated Gouda cheese during 2 h of brining at 13 degrees C resulted in production of small gas holes during ripening. In Gouda cheese slurry stored at 13 degrees C, three C. tyrobutyricum strains plus one of three C. sporogenes strains germinated in the slurry with no added salt. Of three C. tyrobutyricum strains stored at 13 degrees C in slurries with higher water-phase salt concentrations of 2.4 and 3.6%, two strains and one strain germinated, respectively. No germination of spores was detected in any cheese slurry stored at 5 or 8 degrees C. Milk centrifugation, increased percent water-phase salt, absence of spores in brine, and decreased ripening temperature are all potentially important measures against gas production by Clostridium spp. in Gouda cheese.     

Effect of a nisin-producing lactococcal starter on the late blowing defect of cheese caused by Clostridium tyrobutyricum

Clostridium tyrobutyricum causes swelling, cracks and off-flavours of cheeses (late blowing defect, LBD) due to butyric acid fermentation. To control this spoilage bacterium, we investigated the use of nisinogenic Lactococcus lactis subsp. lactis INIA 415 as starter in cheeses contaminated with C. tyrobutyricum spores. Control cheese made with spores showed LBD after 14 days of ripening. However, in cheese made with the bacteriocin producer and spores, in which (unlike control cheese) bacteriocin activity was detected throughout ripening, LBD occurred after 21 days. At this stage, level of lactic acid was 1.22-fold higher (P < 0.01) and concentrations of propionic and butyric acids were 2.15- and 2.32-fold, respectively, lower (P < 0.01) in cheese made with the nisin producer than in control cheese, according to the less pronounced spoilage symptoms showed by the former cheese. The bacteriocin producer delayed the appearance of LBD, although it cannot arrest completely C. tyrobutyricum growth.     

Accumulation of short n-chain ethyl esters by esterases of lactic acid bacteria under conditions simulating ripening Parmesan cheese

EstA from Lactobacillus helveticus CNRZ32 (Lbh-EstA), EstB, and EstC from Lactobacillus casei LILA, and EstA from Lactococcus lactis MG1363 (Lcl-EstA) were evaluated for their ability to accumulate esters in a model system simulating Parmesan cheese ripening conditions (10 degrees C, 2 to 3% NaCl, pH 5.4 to 5.5, aw = 0.850 to 0.925) using Capalase K from kid goat as a positive control. All of the LAB esterases and Capalase K mediated the accumulation of esters in the model system in an enzyme specific manner, which was influenced by a, and selectivity for fatty acid chain-length. In general, enzyme mediated accumulation of ethyl esters was higher at aw values of 0.850 and 0.900 than at aw of 0.925, demonstrating that aw is a critical parameter influencing ester accumulation. The substrate selectivity of esterases, aw, and enzyme type may be important factors in the development of fruity flavors, as evidenced by results in this model system simulating Parmesan cheese ripening conditions.     

应用回归正交法确定豆酪生产条件

以大豆为原料,通过嗜乳杆菌乳酸菌和毛霉联合发酵制作新型大豆发酵制品——豆酪的最佳工艺条件。结果表明豆酪的消费者总体喜爱程度与嗜乳杆菌乳酸菌及毛霉的接种量有关,而在3%~9%的范围内与加盐量无关。当嗜乳杆菌乳酸菌的接种量在5.5%~6.5%之间,毛霉接种量在1%~1.8%之间时,豆酪的消费者总体喜爱程度得分在8~10之间。     

Rapid detection and identification of Streptococcus macedonicus by species-specific PCR and DNA hybridisation

The aim of this study was to develop a simple and specific method for the rapid detection and identification of Streptococcus macedonicus. The method was based on polymerase chain reaction (PCR) using species-specific primers derived from the 16S rRNA gene. Specific identification was proven on seven S. macedonicus strains, while 16 strains belonging to different lactic acid bacteria species were tested negative. The PCR assay was capable of detecting 100 pg of S. macedonicus DNA, and it was also efficient on single colonies of the bacterium. Furthermore, the same bacterial strains were used for the specificity evaluation of a S. macedonicus species-specific probe. Neither species-specific PCR nor DNA hybridisation experiments could differentiate Streptococcus waius from S. macedonicus, due to the identity of the 16S rRNA gene of the two species, indicating high phylogenetical relatedness. This was further confirmed by the comparative sequence analysis of the 16S-23S rRNA intergenic regions. It was thus clearly demonstrated that S. waius, recently described as a novel Streptococcus species, is phylogenetically identical to S. macedonicus.     

Rapid assessment of the physiological status of Streptococcus macedonicus by flow cytometry and fluorescence probes

Flow cytometry in combination with fluorescence probes was applied to rapidly assess the physiological status of Streptococcus macedonicus ACA-DC 198, a newly described member of the lactic acid bacteria group with technologically important features (e.g. lantibiotic production). A sonication procedure was developed for disaggregating typical streptococci chains in order to optimize cell preparations for single cell analysis. Single stained live and dead populations of S. macedonicus cells were clearly resolved based on membrane potential by bis-oxonol [DiBAC(4)(3)], membrane integrity by Propidium Iodide (PI) and enzymatic activity as well as membrane integrity by Carboxyfluorescein Diacetate (cFDA). Further, estimation of both live and dead cells by a cFDA/PI two-colour flow cytometric assay showed excellent correlation with the dead cells in the samples (dead(FCM)=0.9945 dead(S)-0.806, R(2)=0.9986 and live(FCM)=-0.978 dead(S)+98.895, R(2)=0.9992). Finally, the assay was applied to study the physiology of S. macedonicus after acid stress. Interestingly, in situ assessment of the physiological status of stressed S. macedonicus cells by flow cytometry and single cell sorting revealed the coexistence of three distinct subpopulations according to their fluorescence labelling behaviour and culturability, representing intact/culturable, permeabilized/dead and potentially injured cells with the latter exhibiting both metabolic activity and membrane permeabilization as well as decreased culturability.     

Quality and ripening changes of Ras cheese made by direct acidification

Trials were carried out to produce Ras cheese of good quality without the use of starter. Cheese was made from pasteurized cow's milk acidified with lactic acid or citric acid to pH 5.8 alone or coupled with mixing the curd with glucono δ lactone (4.5 g/kg curd). Control cheese was made from milk ripened with a starter culture of S. lactis. Resultant cheeses showed poor body and texture, weak flavour intensity and low levels of soluble nitrogen compounds and free volatile fatty acids. Incorporation into the cheese curd of mixtures containing Fromase 100 (fungal protease) and Piccantase B (fungal lipase) or Fromase 100 and Capalase K (animal lipase) enhanced flavour intensity, improved body characteristics and accelerated the formation of both soluble nitrogen compounds and free volatile fatty acids. The organoleptic properties of the experimental cheeses with added enzymes were comparable to those of the control cheese.     

Formation of methional by Lactococcus lactis IFPL730 under cheese model conditions

The present work describes the capacity of Lactococcus lactis to produce methional and other sulphur compounds derived from methionine (Met) in a cheese model system. Cheese slurries were prepared from pasteurized ewes' skimmed milk, chemically acidified with glucono-'-lactone and homogenized aseptically adding Met, !-ketoglutarate, pyridoxal 5'-phosphate, thiamine pyrophosphate and NaCl. Slurries were incubated at 12 °C for 14 days with cellular suspensions of L. lactis IFPL359, L. lactis IFPL730 and L. lactis NCDO763 in different combinations. Slurries added with resting cells and the intracellular fraction from L. lactis IFPL730 showed the highest production of methional at the outset of incubation, which decreased during incubation along with a concomitant increase in 3-methylthiopropanol. The sensorial analysis of slurries indicated a characteristic methional aroma (cooked potato-like) in samples containing 4-methylthio-2-ketobutyrate and the intracellular fraction from L. lactis IFPL730. As incubation proceeded, the intensity of methional aroma decreased but samples were judged by the panel tasters as developing a cheese-like flavour.     

Inhibition of Clostridium perfringens by Naphthoquinones

The minimum inhibitory concentrations (MICs) of several naphthoquinones (NQ) were determined in the presence or absence of nitrite (NO₂^?) against various strains of Clostridium perfringens. In fluid thioglycollate medium, MICs ranged from 70–100 ppm for 2-methyl-1,4-NQ (menadione), 200–280 ppm for 1,4-NQ, 180–250 ppm for 1,2-NQ, >500 ppm for several water-soluble derivatives and 100–300 ppm for NO₂^?. Using a type B strain in homogenized meat medium, MICs were 670 ppm for menadione, 620 ppm for 1,4-NQ and 770 ppm for NO₂^?. Nitrite, menadione and 1,4-NQ exhibited comparable and additive rather than synergistic inhibition. Some NQ compounds may have potential as partial nitrite substitutes subject to safety evaluation.     

Fate of Listeria innocua during production and ripening of smeared hard cheese made from raw milk

The fate of 2 different Listeria innocua strains was analyzed during the production and ripening of smeared raw milk Greyerzer cheese (Gruyère). These strains were used as surrogates for the pathogenic Listeria monocytogenes, as they are physiologically very similar. Bacterial cells were added to the cheese milk at levels of 10⁵ cfu/mL. During the first 24 h of cheese making, the number of the test strains decreased to a level of below 10² cfu/g. Obviously, the cooking temperature of 56°C and the subsequent slight temperature decrease to 50°C within 70 min contributed to a distinct reduction of Listeria counts. The counts in the cheese cores did not exceed 10³ cfu/g within 12 wk of cheese ripening and Listeria was not detectable after 24 wk. In contrast to the cores of the cheeses of the 4 batches in this study, their rinds always contained a high listerial load of approximately 10⁶ to 10⁸ cfu/g throughout the entire ripening period. The smeared surface showed an increase of pH to alkaline values, corresponding to smear microbiota development. Coryneforms and Staphylococcus counts were stable at >10⁷ cfu/cm² over 175 d, whereas yeast counts decreased to about 10⁵ cfu/cm² at the end of ripening. The study shows that the smear culture had no noticeable anti-listerial potential. When removing the rind or portioning such smeared cheese loaves with a cutting device, a postprocess contamination of the core might occur, thus presenting a major hygienic risk.     

Inhibition of Clostridium sporogenes growth in mascarpone cheese by co-inoculation with Streptococcus thermophilus under conditions of temperature abuse

The ability of a biological control system to inhibit the outgrowth of Clostridium sporogenes spores during storage of mascarpone cheese under temperature-abuse conditions was investigated. Challenge studies were carried out on mascarpone cheese artificially contaminated with spores of C. sporogenes (10 cfu g⁻¹), and with or without the coinoculum of a Streptococcus thermophilus strain (10⁵cfu g⁻¹). During storage at 4, 12, and 25°C, the outgrowth of clostridia spores, the growth of S. thermophilus, and the pH changes were evaluated at 10, 20, 30, and 40 days. In mascarpone cheese stored at 4° and 12°C, S. thermophilus and C. sporogenes did not show any growth. The initial pH (6·14) of the product also remained unchanged. During storage at 25°C S. thermophilus grew up to about 10⁷cfu g⁻¹after 10 days, resulting in a pH decrease of mascarpone cheese to values close to 4·5. The cell number decreased progressively during storage reaching values near to 10¹cfu g⁻¹after 40 days, whereas product acidity remained constant. C. sporogenes, when inoculated alone, also grew at 25°C. The cell number increased to levels of about 10⁷cfu g⁻¹after 20–40 days of storage according to the different mascarpone cheese lots used. No growth was found when C. sporogenes was co-inoculated in mascarpone cheese with S. thermophilus and stored at 25°C. The study on the behaviour of C. sporogenes, known as a non-toxigenic variant of Clostridium botulinum, allowed us to obtain useful information for setting up an effective biological control system to inhibit growth of the toxigenic species as well. The use of an additional barrier, besides refrigerated storage, may help to maintain the safety of mascarpone cheese in the event it was exposed to elevated temperatures.