Whey Starter Research Articles

Behavior of Lactococcus lactis subsp. lactis biovar. diacetylactis in a Four Lactococcus Strain Starter during Successive Milk Cultures

Cheeses are frequently made with natural whey starters (NWS). The whey from the previous cheese making is cultured and used for the next day. This practice, although essential for the development of typical sensory characteristics, can sometimes lead to acidification defects. In this work, the ability of Lactococcus lactis subsp. lactis biovar. diacetylactis to dominate over the other lactic acid bacteria (LAB) was tested in a controlled system as a possible explanation for these acidification breakdowns. A starter made of two Lc lactis subsp. lactis strains (LL), one Lc lactis subsp. cremoris strain (LC), and one Lc lactis subsp. lactis biovar. diacetylactis (LD) was added to sterilized milk. After incubation, the whey was removed and used to re-seed sterilized milk, the next day. This process was made during a five and twelve days' period. During the eight first days, the proportion of LD population increased, while the other LAB remained rather stable. Thereafter, LD strains dominated. At the same time, the diversity of LD population diminished considerably. If acidification ability of these LAB is not altered, this simplification was particularly hazardous in case of phages attack. LC and LL behavior was tested in milk containing increasing diacetyl concentrations. As long as diacetyl did not exceed a 5 ppm level — frequently measured in dairy products — its influence was impossible to detect. The selective advantage conferred by the citrate metabolism was proposed as a possible explanation for the LD population dominance. Other potential factors were also examined.

Influence of backslopping on the acidifications curves of “Tomme” type cheeses made during 10 successive days

The use of natural whey starter (NWS), also named “backslopping”, is still widespread for several cheesemaking processes. This practice plays a decisive role in the cheese typicity but can also lead to technological defects. A lack of knowledge exists concerning these problems. In this research, “Tomme” cheese variety is used as a model to study backslopping. A starter made of three characterized lactic acid bacteria (LAB) strains was used on day one. During the 10 following days, the whey was used from one day to the other as NWS. The milk used was pasteurized and re-seeded with identified strains of LAB to mimic the contribution of the raw milk flora. As expected, the pH evolution during molding changed from day to day. The analysis of the compositional data, and among them, moisture in non fat cheese and calcium on non fat dry matter, and the use of a statistical PLS approach allowed to prove that pH fluctuations were simply explained by the evolution of the composition of NWS. These results reinforced the accuracy of the model to study backslopping.

Antibiotic resistance and microbial composition along the manufacturing process of Mozzarella di Bufala Campana

The use of antibiotics as growth promoters in livestock, banned in all EU member states in January 2006, has led to selection of antibiotic resistant strains within environmental bacteria, including gram-positive, non pathogenic bacteria that colonize the GI tract of humans and animals. In Italy and in other Mediterranean countries, fermented foods employing environmental bacteria pre-existing in the raw substrates, rather than industrial starters of defined genotype, represent a significant proportion of cheese and meat products carrying the official PDO designation (Protected Designation of Origin). Our study focused on the microbiological and molecular analysis of lactobacilli and of other lactic acid bacteria (LABs) isolated from the Italian PDO product water buffalo Mozzarella cheese, with the aim of identifying genes responsible for tetracycline, erythromycin and kanamycin resistance. We isolated over 500 LAB colonies from retail products, as well as from raw milk and natural whey starters employed in their production. Microbiological analysis showed that about 50% of these isolates were represented by lactobacilli, which were further characterized in terms of species and strain composition, as well as by determining phenotypic and genotypic antibiotic resistance. To overcome the limits of culture-dependent approaches that select only cultivable species, we have also extracted total DNA from the whole microbiome present in the cheese and investigated the presence of specific antibiotic resistance genes with molecular approaches. Genetic determinants of antibiotic resistance were identified almost exclusively in bacteria isolated from the raw, unprocessed substrates, while the final, marketed products did not contain phenotypically resistant lactobacilli, i.e. displaying MIC values above the microbiological breakpoint. Overall, our results suggest that the traditional procedures necessary for manufacturing of this typical cheese, such as high temperature treatments, lead to a final product with low bacterial counts, lower biodiversity and lack of significant presence of antibiotic resistant lactobacilli.

Parmigiano Reggiano cheese: evolution of cultivable and total lactic microflora and peptidase activities during manufacture and ripening

Parmigiano Reggiano is a Protected Designation of Origin, long-ripened cheese, made from cow’s milk supplemented with natural whey starter, which thus contains a large microbial biodiversity. The aim of this study was to understand the population dynamics of the total lactic microflora throughout the manufacture and ripening of this cheese. Several approaches were combined to determine the quantitative changes in the different bacterial populations during 20 months of ripening of Parmigiano Reggiano cheeses from the same cheesemaking. Total and viable cells were enumerated after fluorescent labeling. Culturable bacteria were enumerated on different plate count agar media, including original media prepared from curd and ripened cheese. Six peptidase activities were quantified in curd and cheese samples free from cells. While the total bacterial cultivable population remained high and similar for the first six months, a decrease in viable starter lactic acid bacteria was observed during the first 48 h. The non-starter lactic acid bacteria populations, initially present in low numbers, began to grow after the brining and remained at high levels (about 107 CFU·g−1) for at least 10 months. During ripening, a strong decrease in the total bacterial population and a marked increase in 4 out of 6 peptidase activities were observed. In the external and internal zones of Parmigiano Reggiano cheese different trends in microbial growth, cell autolysis and peptidase activity were observed. This study gives for the first time a global view of the possible contribution of total, viable, cultivable and lysed bacterial cells throughout the ripening of Parmigiano Reggiano cheese.

Grana Padano cheese whey starters: Microbial composition and strain distribution

The aim of this work was to evaluate the species composition and the genotypic strain heterogeneity of dominant lactic acid bacteria (LAB) isolated from whey starter cultures used to manufacture Grana Padano cheese. Twenty-four Grana Padano cheese whey starters collected from dairies located over a wide geographic production area in the north of Italy were analyzed. Total thermophilic LAB streptococci and lactobacilli were quantified by agar plate counting. Population structure of the dominant and metabolically active LAB species present in the starters was profiled by reverse transcriptase, length heterogeneity-PCR (RT–LH–PCR), a culture-independent technique successfully applied to study whey starter ecosystems. The dominant bacterial species were Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum. Diversity in the species composition allowed the whey cultures to be grouped into four main typologies, the one containing L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus being the most frequent one (45% of the cultures analyzed), followed by that containing only the two lactobacilli (40%). Only a minor fraction of the cultures contained L. helveticus alone (4%) or all the four LAB species (11%). Five hundred and twelve strains were isolated from the 24 cultures and identified by M13-PCR fingerprinting coupled with 16S rRNA gene sequencing. Most of the strains were L. helveticus (190 strains; 37% of the total), L delbrueckii subsp. lactis (90 strains; 18%) and S. thermophilus (215 strains; 42%). This result was in good agreement with the qualitative whey starter composition observed by RT–LH–PCR. M13-PCR fingerprinting indicated a markedly low infra-species diversity, i.e. the same biotypes were often found in more than one culture. The distribution of the biotypes into the different cultures was mainly dairy plant-specific rather than correlated with the different production areas.

Perfil de ácidos grasos libres y características sensoriales de quesos reggianito elaborados con diferentes fermentos

Reggianito is the most important variety among the Argentinean hard cheeses. During its ripening period several biochemical reactions of proteins and fat are responsible for its final sensory characteristics. The fat transformations, in a first step, produce free fatty acids (FFA). In the present work, the FFA profile from C 6:0 to C 18:2 during ripening was evaluated. Reggianito cheeses were produced with a “wild” whey starter and with selected strains of Lactobacillus helveticus isolated from natural whey. Information from FFA profiles was processed by Principal Component Analysis (PCA) and Discriminant Analysis (DA). Despite the fact that FFA levels grew continuously during ripening, no differences were found among cheeses produced with different starters. Sensory analyses did not show differences among the different cheeses produced. All cheeses were of satisfactory sensory quality. Results from this study are important for an increased knowledge of the ripening process of Reggianito cheese, considering the scarce information currently existing on this subject. At the same time, the substitution of a natural whey starter for a selected strain starter appears to be a possible advantage in order to improve and to standardize the quality of the cheese.

Detection and identification ofLactobacillus helveticusbacteriophages by PCR

A PCR protocol for detection of Lactobacillus helveticus bacteriophages was optimized. PCR was designed taking into account the sequence of the lys gene of temperate bacteriophage Phi-0303 and optimized to obtain a fragment of 222 bp using different Lb. helveticus phages from our collection. PCR was applied to total phage DNA extracted from 53 natural whey starters used for the production of Grana cheese and all gave the expected fragment. The presence of actively growing phages in the cultures was verified by traditional tests. Several PCR products of the lys gene were sequenced and aligned. The resulting sequences showed variable heterogeneity between the phages.

Selection and use of autochthonous multiple strain cultures for the manufacture of high-moisture traditional Mozzarella cheese

Lactobacillus plantarum 18A, Lactobacillus helveticus 2B, Lactobacillus delbrueckii subsp . lactis 20F, Streptococcus thermophilus 22C, Enterococcus faecalis 32C and Enterococcus durans 16E were the most acidifying strains within 146 isolates for natural whey starters. The effect of media and temperature on 2 autochthonous multiple strain cultures (AMSI: 18A, 2B, 20F and 22C, 32C and 16E and AMSII: 18A, 2B, 20F and 22C) was studied. Genomic analysis showed a constant cell numbers for AMSII during 16 days of propagation in whey milk. Mozzarella cheese was made by using AMSII, commercial starter (CS) or citric acid (DA). Compared to other cheeses, the DA had a lower level of protein, ash, Ca, free amino acids and a higher level of moisture. Based on confocal laser scanning microscopy analysis, AMSII cheese showed the lowest microstructural variations during the period of storage compared to other cheeses. All the sensory attributes were scored highest for AMSII cheese. ASMII extend the shelf-life to ca. 12−15 days instead of the 5−7 days of traditional high-moisture Mozzarella cheese.

Whey Starter for Grana Padano Cheese: Effect of Technological Parameters on Viability and Composition of the Microbial Community

This work aimed to investigate the effects of thermal treatments and yeast extract addition on the composition of the microbial community of natural whey starters for Grana Padano cheese. Different natural whey starter samples were held at 4°C for 24h (cooling treatment), or at −20°C for 24h (freezing treatment) to evaluate the possibility of conservation, or at 54°C for 1h (heat treatment) to evaluate the effect of the temperature commonly used during curd cooking. Separately, another set of samples was enriched with 0.3, 0.5, and 1.0% (wt/vol) of yeast extract to study its effect on the growth of lactic acid bacteria (LAB) in the starter. The new approach in this study is the use of 2 culture-independent methods: length heterogeneity (LH)-reverse transcription (RT)-PCR and fluorescence microscopy. These techniques allowed us to easily, quickly, and reproducibly assess metabolically active LAB in the control and treated samples. The LH-RT-PCR technique distinguished microorganisms based on natural variations in the length of 16S rRNA amplified by RT-PCR, as analyzed by using an automatic gene sequencer. Fluorescence microscopy counts were performed by using a Live/Dead BacLight bacterial viability kit. The repeatability of LH-RT-PCR showed that this technique has great potential to reveal changes in the microbial community of natural whey starters for Grana Padano cheese. All species showed low sensitivity to cold (4°C). However, after the freezing (−20°C) and heating (54°C) treatments, different behaviors of the species were reported, with significant changes in their viability and relative composition. Heating treatment during curd cooking profoundly affected the viability and composition of the community that remained in the cheese and that consequently modified the microbial population. At the same time, this treatment produced the selection of LAB in whey and could be considered as the first step in natural whey starter production. Addition of yeast extract stimulated the growth of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. lactis to the detriment of Lactobacillus helveticus species. Because the yeast extract altered the microflora balance, whey starter conservation at −20°C and yeast extract addition cannot be suggested as technological innovations.

Population dynamics of lactobacilli in Grana cheese

A survey on the presence, microbial diversity, and population dynamics of lactobacilli in Grana cheese is presented. Evolution of thermophilic rod lactic acid bacteria within the first two days from cheese making and during ripening was different according to different bacterial groups, which were selectively enumerated and identified by molecular methods. Species-specific microbial counts indicated prevalence ofLactobacillus helveticus in both the whey starter and the cheese at moulding, and ofLactobacillus delbrueckii subsp.lactis in cheese after two months of ripening. In more advanced ripening, a decrease of total thermophilic lactobacilli and an increase of mesophilic lactobacilli (mostly belonging toLactobacillus casei/paracasei andLactobacillus rhamnosus) was observed. PCR fingerprinting of lactobacilli, which was performed by PCR-fingerprinting, indicated a marked microbial heterogeneity within theLactobacillus spp. populations, which enabled strain (or group)-specific fingerprints to be observed.

Molecular identification and typing of natural whey starter cultures and microbiological and compositional properties of related traditional Mozzarella cheeses

Cell numbers of presumptive lactic acid bacteria varied markedly between 7 natural whey starter cultures (NWSC) used for producing traditional cows' milk Mozzarella cheeses in the Apulia region of Southern Italy. Taxonomic identification revealed a large diversity at species level, including mesophilic and thermophilic lactobacilli, lactococci, streptococci and enterococci. Randomly Amplified Polymorphic DNA (RAPD-PCR), analysis showed the biodiversity among the strains and, for lactobacilli, some relationships with provenience of the natural starter. Cell numbers of presumptive lactic acid bacteria in the corresponding Mozzarella cheeses were similar or higher than those found in the corresponding NWSC. RAPD-PCR analyses showed that most of the strains in cheese originated from the starter. The gross composition varied markedly between the 7 Mozzarella cheeses and ranged from 53−64% moisture, 17−23% protein, 13−20% fat and 0.50−1.61% salt. The values of pH for several samples were above 6.0. As shown by urea-PAGE of the pH 4.6-insoluble nitrogen fractions, cheese samples were characterized by differences in α S1- and β-casein hydrolysis. Cheeses also differed with respect to secondary proteolysis as shown by Principal Component Analysis (PCA) of data from RP-HPLC of the pH 4.6-soluble, pH 4.6–70% ethanol-soluble and 70% ethanol-insoluble nitrogen fractions. These differences were attributed to the different microbial composition of the NWSC. Strain selection and optimization of a protocol for producing a natural whey starter culture to be used by dairy factories of the Apulia region appears to be a pre-requisite to standardize the major traits distinguishing this cheese variety.

Detection and Characterization of Listeria monocytogenes in São Jorge (Portugal) Cheese Production

Listeria monocytogenes is a foodborne pathogen that can cause serious invasive disease in humans. Because human listeriosis cases have previously been linked to consumption of contaminated cheese, control of this pathogen throughout the cheese production chain is of particular concern. To understand the potential for L. monocytogenes transmission via São Jorge cheese, a Portuguese artisanal cheese variety that bears a Protected Denomination of Origin classification, 357 raw milk, curd, natural whey starter, and cheese samples representative of the production chain of this cheese were collected over one year and tested for the presence of L. monocytogenes and selected physicochemical parameters. Although neither L. monocytogenes nor other Listeria spp. were detected in whey, curd, or cheese samples, 2 of the 105 raw milk samples analyzed were positive for L. monocytogenes. These 2 raw milk isolates represented a ribotype that has previously been linked to multiple human listeriosis outbreaks and cases elsewhere, indicating the potential of these isolates to cause human listeriosis. On average, physicochemical parameters of São Jorge cheese ripened for 4 mo presented values that likely minimize the risk of L. monocytogenes outgrowth during ripening and storage (mean pH=5.48; mean moisture=37.79%; mean NaCl concentration=4.73%). However, some cheese samples evaluated in this study were characterized by physicochemical parameters that may allow growth and survival of L. monocytogenes. Even though our results indicate that raw milk used for São Jorge cheese manufacture as well as finished products is rarely contaminated with L. monocytogenes, continued efforts to control the presence of this pathogen in the São Jorge cheese production chain are urged and are critical to ensure the safety of this product.

Spread and variability of the integrase gene in Lactobacillus delbrueckii ssp. lactis strains and phages isolated from whey starter cultures

To determine the presence, diffusion and variability of the integrase (int) gene in Lactobacillus delbrueckii ssp. lactis isolated from natural whey starters used for the production of Italian hard cheeses. A PCR-based protocol aimed to amplify an internal fragment of the int gene was optimized taking into account phage genome sequences available from public databases. Thirty-seven of the 39 strains tested showed the presence of the putative int gene. Southern blot hybridization experiments confirmed data obtained by PCR. The presence of the putative int gene was observed also in 20 of 23 Lact. delbrueckii ssp. lactis lytic phages isolated from the same starter cultures used to isolate strains. Phylogenetic analysis of partial int gene revealed a high similarity both within and between strain- and phage-derived sequences. Sixty per cent of the int-positive strains resulted inducible with mitomycin C, and two of them released active phage particles. Our preliminary findings seem to suggest that an important number of Lact. delbrueckii ssp. lactis strains associated with the whey starters are lysogenic. Further contribution to obtain a clearer picture of the complex relationship between thermophilic lactic acid bacteria phage and host in whey starters for Italian, hard-cooked cheeses.

Multilocus restriction typing: A tool for studying molecular diversity within Lactobacillus helveticus of dairy origin

Forty-two Lactobacillus helveticus strains isolated from two whey starter cultures and three cheeses were typed by multilocus restriction typing (MLRT), analyzing the restriction fragment-length polymorphism of PCR products generated from eight loci of housekeeping genes: β-galactosidase, lactose permease, UDP-galactose-4 epimerase, β-galactosidase transcriptional regulator, D-lactate dehydrogenase, peptidase N, stress-inducible protein and S-layer protein. MLRT analysis indicated wide strain heterogeneity. Strains were grouped according to their source of isolation. The low intra-species polymorphism detected in the 16S rRNA gene did not allow grouping of the strains with the same sensitivity reached by MLRT of protein-coding genes. This is the first demonstration of MLRT as a technique for the discrimination of Lb. helveticus strains; this technique is a promising tool to evaluate genetic diversity of related strains from different ecological niches.

Fluorescence microscopy for studying the viability of micro-organisms in natural whey starters

The aim of this work was to study the viability and cultivability of microbial populations of different natural whey starters and to evaluate their resistance to thermal treatments (such as exposure to high or low temperatures). Twenty-three natural whey starters for Grana Padano cheese were investigated and subsequently pH measurement, plate count agar using Man-Rogasa-Sharpe (MRS) pH 5.4 agar and whey agar medium (WAM) were performed using these samples. LIVE/DEAD BacLight bacterial viability kit was used. Total count and viability of all the 23 samples were high and similar to each other (CV 20%). However, the cultivable population was lower in terms of cfu ml(-1) and number of cells per millilitre than the viable fraction and highly variable, although its count value was higher in WAM than in MRS pH 5.4. The heating (60 degrees C for 5 min and 54 degrees C for 1 h) and freezing (-20 and -80 degrees C) treatments affected the cultivability and viability of the microbial population. This study demonstrated the effectiveness of LIVE/DEAD BacLight bacterial viability kit, which has already been used to evaluate bacterial populations, in investigating microbial viability in a complex ecosystem such as a natural whey starter. The aim of this study was to quantify the presence of damaged nonviable bacterial cells in natural whey starters. The Thoma Glass is a useful method to obtain fluorescence microscopy counts to evaluate the technological performance of natural whey starters.

Cultivability ofStreptococcus thermophilusin Grana Padano cheese whey starters

The application of a culture-independent approach, that of reverse transcriptase-length heterogeneity-PCR coupled with epifluorescence microscopy, allowed us to observe that Streptococcus thermophilus is metabolically active, but only partially cultivable in Grana Padano cheese whey starters. A short preincubation of the starters in sterile skimmed whey was followed by cultivation in sterile skimmed whey-enriched M17. This procedure restored the cultivability of S. thermophilus and enabled us to detect S. thermophilus at ranges (10(7)-10(8) CFU mL(-1)) which have rarely been reported in these cultures. The use of cheese whey as a cultivation-revitalization substrate can be useful to obtain an unbiased picture of the microbial composition of whey starters for Grana Padano cheese, thus avoiding an underestimation of S. thermophilus in these cultures.

Detection and identification of Lactobacillus delbrueckii subsp. lactis bacteriophages by PCR

A sensitive PCR method amplifying an internal fragment of the major tail protein gene was developed to detect Lactobacillus delbrueckii subsp. lactis lytic bacteriophages in undefined, thermophilic whey starters used in Italy for production of Grana and Provolone cheeses. PCR was applied to several lytic Lb. delbrueckii subsp. lactis bacteriophages, which were highly diverse according to restriction analysis and phage host range. PCR detected the presence of phages in two out of 11 cultures, when applied to whey starters for Grana Padano cheese sampled from different cheese plants. The presence of actively growing phages in infected cultures was confirmed by traditional test. The PCR method proved to be useful to screen for the presence of Lb. delbrueckii subsp. lactis phages in thermophilic whey starters.

Employment of autochthonous microflora in Pecorino Sardo cheese manufacturing and evolution of physicochemical parameters during ripening

The isolation and identification of lactic acid bacteria (LAB) from raw ewes' milk and traditional Pecorino Sardo cheese made from this milk without the addition of starter culture was carried out to define the autochthonous lactic microflora present in milk and the evolution of LAB during cheese ripening. Isolation of 275 strains belonging to different Lactococcus, Lactobacillus, Streptococcus and Enterococcus species was achieved. Coccal-shaped LAB were found to predominate during cheese fermentation, while lactobacilli were preponderate during the latter phase of ripening. The technological selection of a total of 174 LAB strains belonging to the species Lactococcus lactis, Streptococcus thermophilus, Lactobacillus helveticus and Lb. casei allowed an experimental starter to be prepared, in which a potentially probiotic species, Lb. casei was used. The suitability of the autochthonous starter culture was tested in cheese-making trials, using thermised ewes' milk, by comparing experimental Pecorino Sardo cheese with a control cheese produced at industrial scale using a whey starter culture from previous batches of manufacture. In particular, microbiological and physicochemical parameters were determined over 210 days of cheese ripening. Although sensory evaluation did not show any significant difference between experimental and control Pecorino Sardo cheeses, the use of the selected autochthonous starter allowed the production of experimental cheese with a significantly higher level of free amino acids, in particular essential amino acids, in comparison with the Pecorino Sardo control cheeses.

Free fatty acid profiles of Reggianito Argentino cheese produced with different starters

Abstract Reggianito Argentino cheeses were made at pilot plant scale using a natural whey starter (control) and different combinations of strains of selected Lactobacillus helveticus (experimental cheeses). Free fatty acids (FFAs) were extracted from cheese samples at different ripening times, and FFAs from C 6:0 to C 18:2 were analysed by gas chromatography. The characteristics of the chromatographic profiles were studied by principal components and linear discriminant analysis were performed. Levels of individual FFAs from caproic (C 6:0 ) to linoleic (C 18:2 ) acids increased significantly ( p 0.05 ) during ripening in control and experimental cheeses. Palmitic (C 16:0 ) and oleic (C 18:1 ) acids were the most abundant FFAs throughout ripening in all cheeses. No significant differences were found between FFA profiles of control and experimental cheeses. The possibility of using selected strains of Lactobacillus to replace traditional “natural whey starter” in the production of the cheese is discussed.

Evaluation of bacterial communities belonging to natural whey starters for Grana Padano cheese by length heterogeneity-PCR.

To detect bacteria present in controlled dairy ecosystems with defined composition by length-heterogeneity (LH)-PCR. LH-PCR allows to distinguish different organisms on the basis of natural variations in the length of 16S rRNA gene sequences. LH-PCR was applied to depict population structure of the lactic acid bacteria (LAB) species recoverable from Grana Padano cheese whey starters. Typical bacterial species present in the LAB community were evidenced and well discriminated. Small differences in species composition, e.g. the frequent finding of Streptococcus thermophilus and the constant presence of thermophilic lactobacilli (Lactobacillus helveticus, Lact. delbrueckii subsp. lactis/bulgaricus and Lact. fermentum) were reliably highlighted. Specificity of LH-PCR was confirmed by species-specific PCR from total DNA of the cultures. LH-PCR is a useful tool to monitor microbial composition and population dynamics in dairy starter cultures. When present, non-dominant bacterial species present in the whey starters, such as Strep. thermophilus, can easily be visualized and characterized without isolating and cultivating single strains. A similar approach can be applied to more complex dairy ecosystems such as milk or cheese curd. Community members and differences in population structure of controlled dairy ecosystems such as whey starters for hard cheeses can be evaluated and compared in a relative easy, fast, reliable and highly reproducible way.