Screening and selection of Lactobacillus strains for use as adjunct cultures in production of semi-hard cheese

Thirty-three Lactobacillus strains were tested as adjuncts in a cheese model system. Eighteen strains originated from cheese (nine Lactobacillus spp. and nine Lb. paracasei/casei) and 15 from human intestinal mucosa (11 Lb. rhamnosus; three Lb. paracasei; one Lb. plantarum). Model cheeses weighing 120 g were made of cheese grains from full-scale production of washed curd semi-hard cheese (Herrg?rd). The model system was reproducible and similar to full-scale production with respect to moisture, salt content, pH and microbial flora. The model cheeses were sampled for aerobic and anaerobic plate count and viable counts of Lactobacillus and Lactococcus. The presence of adjuncts in the model cheeses was confirmed by typing isolates with Randomly Amplified Polymorphic DNA (RAPD). The sensory properties of model cheeses were described. In a first trial 23 of the 33 adjuncts were re-isolated from the corresponding model cheeses after 9 or 13 weeks. Adjuncts of Lb. paracasei were re-isolated more frequently than adjuncts of Lb. rhamnosus. Nine strains were selected, on the basis of their ability to grow and be a dominating part of the microflora of model cheese with interesting sensory properties. These strains were further studied together with two commercial cultures. The sensory influences on model cheeses of six of the adjuncts were confirmed, and flavour scores were in the range of 2.9-7.1 for model cheeses with different adjuncts while the control had a flavour score of 5.6 (0-10 scale). Survival and growth of seven out of the nine strains correlated with the results of the first trial. Growth and influence on flavour of four adjunct cultures were confirmed in experimental cheese manufactured in a 400-1 open vat.     

Accelerated ripening of Caciocavallo Pugliese cheese with attenuated adjuncts of selected nonstarter lactobacilli

The nonstarter lactic acid bacteria Lactobacillus plantarum CC3M8, Lactobacillus paracasei CC3M35, and Lactobacillus casei LC01, previously isolated from aged Caciocavallo Pugliese cheese or used in cheesemaking, were used as adjunct cultures (AC) or attenuated (by sonication treatment) adjunct cultures (AAC) for the manufacture of Caciocavallo Pugliese cheese on an industrial scale. Preliminary studies on the kinetics of growth and acidification and activities of several enzymes of AAC were characterized in vitro. As shown by the fluorescence determination of live versus dead or damaged cells and other phenotype features, attenuation resulted in a portion of the cells being damaged and a portion of the cells being capable of growing with time. Compared with the control cheese (without adjunct cultures) and the cheese with AAC, the addition of AC resulted in a lower pH after manufacture, which altered the gross composition of the cheese. As shown by plate count and confirmed by random amplification of polymorphic DNA-PCR, the 3 species of nonstarter lactobacilli persisted during ripening but the number of cultivable cells varied between AC and AAC. Slight differences were found between cheeses regarding primary proteolysis. The major differences between cheeses were the accumulation of free amino acids and the activity levels of several enzymes, which were highest in the Caciocavallo Pugliese cheeses made with the addition of AAC. As shown by triangle test, the sensory properties of the cheese made with AAC at 45d did not differ from those of the control Caciocavallo Pugliese cheese at 60d of ripening. In contrast, the cheese made with AC at 45d differed from both the Caciocavallo Pugliese cheese without adjuncts and the cheese made with AAC. Attenuated adjunct cultures are suitable for accelerating the ripening of Caciocavallo Pugliese cheese without modifying the main features of the traditional cheese.     

Effect of lactobacillus adjunct cultures on the microbiological and physicochemical characteristics of Roncal-type ewes'-milk cheese

The effect of two different experimental adjunct cultures composed of native facultatively heterofermentative lactobacilli (FHL) on the development of various groups of micro-organisms in Roncal-type ewes' milk cheese was studied. Four cheese batches were manufactured from raw milk (C), pasteurized milk (P), pasteurized milk and an adjunct culture of Lactobacillus paracasei (PP); and pasteurized milk and adjunct culture of Lactobacillus paracasei plus Lactobacillus plantarum (PPP). Retention of the two adjunct cultures in the cheeses was good, and population levels remained constant at around 10(7) cfu g(-1) of cheese throughout ripening. Levels of Enterobacteriaceae and enterococci fell off more abruptly in the batches made with the Lactobacillus adjunct cultures, suggesting competition between the added lactobacilli and those groups of micro-organisms. The inhibitory effect was greater for the adjunct culture composed of L. paracasei plus L. plantarum. Lactococcal levels were higher in the batches made with added FHL, which may be indicative of a synergistic effect between these two groups.     

Genetic diversity, dynamics, and activity of Lactobacillus community involved in traditional processing of artisanal Manchego cheese

A total of 248 strains of predominant lactobacilli isolated during the manufacture and ripening of artisanal Manchego cheeses obtained from two dairies were obtained and the genetic diversity of 197 investigated using random amplified polymorphic DNA (RAPD-PCR). 51 isolates could not be lysed and were therefore not genotyped. Forty-two distinct RAPD patterns, grouped in six major clusters at a similarity level of 54%, were obtained. Phenotypic characterization of isolates enabled their assignment to the species L. plantarum, L. brevis, L. paracasei subsp. paracasei, L. fermentum, L. pentosus, L. acidophilus and L. curvatus. In samples from both dairies, the species L. plantarum, L. brevis and L. paracasei subsp. paracasei dominated during ripening. Three genotypes showed excellent physiological characteristics and were therefore proposed as adjunct cultures for Manchego cheese manufacture.     

Cheddar干酪附属发酵剂筛选及其应用

为了筛选能够促进Cheddar干酪成熟,从泡菜、腐乳及不同产地的Cheddar中筛选具有高肽酶活力和自溶度的乳杆菌作为Ched-dar干酪附属发酵剂,并应用于Cheddar干酪的制作中.结果表明,从美国Cheddar干酪中筛选出1株Lactobacillus plantarum OD具有较高的肽酶活力和自溶度,运用于Cheddar千酪的制作中能显著提高其游离氨基酸浓度,改善其风味,有利于加快干酪的成熟.     

Indigenous raw milk microbiota influences the bacterial development in traditional cheese from an alpine natural park

Nostrano di Primiero is a 6-month ripened cheese produced from raw milk collected in the Paneveggio-Pale di San Martino Natural Park area in the Italian Dolomites. In summer, this cheese is made using milk collected from two different areas, Passo Rolle and Vanoi, in the Paneveggio Natural Park. During the experiment, the milk from the two areas was separately processed, and cheeses were made in the same cheese factory using the same technological process. The microbiota of raw milk and cheeses of the two areas was isolated and the dominant population was monitored by RAPD analysis and identified by 16S rRNA sequence. The milk of the Passo Rolle area was mainly composed of mesophilic strains, thermophilic Streptococcus thermophilus, and low amounts of enterococci were also found; the milk of the Vanoi area was dominated by mesophilic microbiota mostly Lactococcus lactis ssp. cremoris and ssp. lactis and Lactobacillus paracasei ssp. paracasei. The plating of the natural starter culture revealed the presence of a relevant community of thermophilic cocci and lower amounts of enterococci. The dynamic population analysis showed the importance of the natural starter culture in the first 2 days of cheese ripening in both cheeses. Moreover, the large biodiversity observed in the raw milks was also detected in the cheeses during ripening. The Vanoi cheese was dominated by Enterococcus faecium and Streptococcus macedonicus in the first two days and mesophilic 21 Lb. paracasei ssp. paracasei became the most represented population after 15 days of ripening. In the first few days, the Rolle cheese was characterized by being mainly composed of thermophilic S. macedonicus and S. thermophilus and secondarily by mesophilic cocci. During ripening, the microbiota composition changed, and at 15 days, mesophilic lactobacilli were the dominant population, but later, this was mainly composed of mesophilic cocci and lactobacilli. The taxonomical identification by 16S rRNA sequence confirmed a large biodiversity related to raw milk microbiota and only five strains of S. macedonicus, Lactobacillus plantarum, 21 Lb. paracasei ssp. paracasei, Lactobacillus fermentum and E. faecium were detected in both cheeses.     

Ripening of cheddar cheese with added attenuated adjunct cultures of lactobacilli

We made Milled curd Cheddar cheese with Lactococcus starter and an adjunct culture of Lactobacillus helveticus I or Lactobacillus casei T subjected to different attenuation treatments: freeze shocking (FS), heat shocking (HS), or spray drying (SD). Proteolysis during cheese ripening (0 to 6 mo), measured by urea-PAGE and water-soluble nitrogen, indicated only minor differences between control and most adjunct-treated cheeses. However, there were significant differences in the effect of Lactobacillus adjuncts on the level of free amino nitrogen in cheese. Cheeses made with FS or HS Lb. helveticus adjunct exhibited significantly greatest rates of free amino group formation. Lipolysis as measured by total free fatty acids was consistently highest in adjunct-treated cheeses, and FS Lb. casei-treated cheeses showed the highest rate of free fatty acid formation followed by FS Lb. helveticus treated cheeses. Mean flavor and aroma scores were significantly higher for cheeses made with Lb. helveticus strain. Freeze-shocked Lb. helveticus-treated cheeses obtained the highest flavor and aroma scores. Sensory evaluation indicated that most of the adjunct-treated cheeses promoted better texture and body quality.     

Characterization of the lactic acid bacteria in ewe's milk and cheese from northwest Argentina

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.     

Influence of autochthonous Argentine goat lactobacillus in ripening of slurry cheese models

Three indigenous lactobacilli isolated from autochthonous Argentinean goat cheeses were evaluated in models of ripening of semihard goat cheeses made under microbiologically controlled conditions. Batches A, B and C were manufactured with single strain of Lactobacillus plantarum ETC17, Lactobacillus rhamnosus ETC14 and Lactobacillus casei ETC19, respectively. Lactobacilli counts showed higher values on batch C until ripening on day 18. Chemical composition did not show differences between batches. Batch C showed a higher decrease in pH on day 1, higher liberation of nitrogen water-soluble cheese extracts and higher free amino acids content up to day 18. Amino acid distribution was analysed by studying the main components.     

Isolation, characterization, and influence of native, nonstarter lactic acid bacteria on Cheddar cheese quality

To determine whether adventitious nonstarter lactic acid bacteria (NSLAB) might affect cheese flavor and quality, we studied a population of NSLAB present in 30 premium quality Cheddar cheeses (3-mo ripened) produced at a commercial facility in the United States. DNA fingerprinting analysis with a sensitive strategy for arbitrary priming polymerase chain reaction showed that 75 isolates corresponded to at least 18 distinct nonstarter organisms. According to ribotype database comparisons of representatives from the 18 groups, 9 matched Lactobacillus (closest to paracasei species), 8 matched Streptococcus thermophilus, and 1 matched to a Lactococcus species. This finding indicated that among the 75 NSLAB isolates, Lactobacillus made up 64%, S. thermophilus 32%, and Lactococcus 4%. Isolates representing 11 NSLAB groups were characterized for protease, peptidase, and diacetyl production. Based on this phenotypic analysis, two Lactobacillus isolates were evaluated as adjuncts in Cheddar cheese. All of the NSLAB identified from the adjunct cheese at 3 mo by DNA fingerprinting consisted of the adjunct lactobacilli, showing that the adjunct strains predominated throughout the early stages of ripening. The impact of adjunct lactobacilli was evident after 6 mo when free amino acids significantly increased and sensory scores improved in adjunct cheese as compared with a control cheese. The largest impact was found in adjunct cheese containing a blend of both lactobacilli strains. These results show that certain adventitious NSLAB positively contribute to flavor development.     

Influence of adjunct use and cheese microenvironment on nonstarter bacteria in reduced-fat cheddar-type cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7 degrees C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 10(4) cfu/ml did not completely suppress "wild" NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.     

Isolation of lactic acid bacteria from Lighvan cheese, a semihard cheese made from raw sheep milk in Iran

The lactic acid bacteria contributing to Lighvan cheese ripening during the different stages of production were investigated. Isolated strains from different culture media were identified phenotypically to species and subspecies level. In total, 413 strains were isolated from raw milk, 1-day-old cheese and fully ripened cheese. The most abundant species belonged to Enterococcus faecium (87 isolates), Lactococcus lactis ssp. lactis (68 isolates), Enterococcus faecalis (55 isolates) and Lactobacillus plantarum (48 isolates). E. faecium, Lc. lactis and Lb. plantarum were the predominantly isolated strains from ripened cheese. Therefore, they may contribute considerably to the aroma and flavour development of Lighvan cheese.     

Microflora of Feta cheese from four Greek manufacturers

The components of the microflora of four Feta cheeses, produced by different Greek manufacturers, were determined by culture dependent and independent techniques. Isolates from microbiological media were first grouped by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and then representatives of each DGGE group were sequenced for identification purposes. DNA and RNA, extracted directly from the cheese, were subjected to PCR-DGGE. Moreover, Feta cheeses were subjected to FISH analysis in order to identify viable bacterial populations. The microbial ecology, as represented by the Lactic Acid Bacteria (LAB) and yeast populations, was different for the four cheeses. The main LAB species isolated were Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus coryniformis and Lactobacillus fermentum. However, some inconsistencies were observed between the results obtained with the culture dependent and the culture independent approach. In the case of the yeasts, the results obtained by PCR-DGGE compared very well with those obtained by the conventional microbiological analysis and the main species found were Kluyveromyces lactis, Pichia fermentans and C. zeylanoides. FISH analysis highlighted viable but not culturable populations of Streptococcus thermophilus and Lactococcus spp. RAPD-PCR performed on the L. plantarum isolates revealed a cheese specific distribution and a temperature dependent clustering.     

Diversity and dynamic of lactic acid bacteria strains during aging of a long ripened hard cheese produced from raw milk and undefined natural starter

The aim of this study was to explore diversity and dynamic of indigenous LAB strains associated with a long ripened hard cheese produced from raw milk and undefined natural starter such as PDO Grana Padano cheese. Samples of milk, curd, natural whey culture and cheeses (2nd, 6th, 9th and 13th months of ripening) were collected from 6 cheese factories in northern Italy. DNA was extracted from each sample and from 194 LAB isolates. tRNA^Ala-23S rDNA-RFLP was applied to identify isolates. Strain diversity was assessed by (GTG)5 rep-PCR and RAPD(P1)-PCR. Finally, culture-independent LH-PCR (V1–V2 16S-rDNA), was considered to explore structure and dynamic of the microbiota. Grana Padano LAB were represented mainly by Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus helveticus and Pediococcus acidilactici, while the structure and dynamic of microbiota at different localities was specific. The strength of this work is to have focused the study on isolates coming from more than one cheese factories rather than a high number of isolates from one unique production. We provided a valuable insight into inter and intraspecies diversity of typical LAB strains during ripening of traditional PDO Grana Padano, contributing to the understanding of specific microbial ecosystem of this cheese.     

Influence on cheese proteolysis and sensory characteristics of non-starter lactobacilli strains with probiotic potential

The contribution to cheese proteolysis and sensory profile of four potentially probiotic non-starter lactobacilli strains was assessed in two different models: soft and semi-hard cheeses. All the strains were able to grow in both types of cheese, where they maintained high levels during ripening. Overall, adjunct cultures of Lactobacillus rhamnosus showed the strongest influence in peptidolysis, which was verified by changes in peptide profiles and increase of free amino acids concentration. Nevertheless, some cheeses treated with L. rhamnosus also showed post-acidification during ripening and decreased sensory characteristics compared to controls. Cheeses with adjunct cultures of Lactobacillus casei I90 and Lactobacillus plantarum I91 exhibited, in general, an intermediate level of peptidolysis between control and L. rhamnosus-added cheeses; their sensory characteristics were preserved or improved, and they did not show any defects. Adjunct cultures showed similar trends in both models, confirming L. casei I90 and L. plantarum I91 as the most performing adjunct cultures for cheese-making among the strains.     

Diversity of lactic acid bacteria isolated from AOC Salers cheese

The objective of this work was to describe the diversity of lactic acid bacteria in traditional raw milk Salers cheeses at the species and strain levels. The characterization of 381 strains isolated during ripening and various strain collections was investigated using physiological analysis and molecular techniques: Rep-PCR, species and genus specific amplifications and the sequence analysis of 16S rDNA for strain typing and taxonomic identification. The strains belonged to Lactobacillus plantarum, Lactobacillus paracasei, Lactococcus lactis, Lactococcus garviae, Enterococcus faecalis, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Streptococcus salivarius, Streptococcus millieri, Streptococcus macedonicus and Pediococcus pentosaceus. A wide phenotypic and genomic heterogeneity was observed within the different species (Lactobacillus plantarum, Lactobacillus paracasei and Leuconostoc mesenteroides) according to the origin and the time of ripening. The natural microflora was different from strain collection and each method must be combined to identify and characterize natural microflora. This study revealed the low selectivity of selective media used for the isolation of different groups of lactic acid bacteria except the Facultatively Heterofermentative lactobacilli medium selecting mesophile lactobacilli and SB medium selective for Enterococcus. The study reveals, for the first time, the microbial lactic acid bacteria community of Salers cheese and its diversity. A better knowledge of microbial flora will be useful to improve understanding of sensory quality of cheeses.     

Lactobacillus casei strains isolated from cheese reduce biogenic amine accumulation in an experimental model

Tyramine and histamine are the biogenic amines (BAs) most commonly found in cheese, in which they appear as a result of the microbial enzymatic decarboxylation of tyrosine and histidine respectively. Given their toxic effects, their presence in high concentrations in foods should be avoided. In this work, samples of three cheeses (Zamorano, Cabrales and Emmental) with long ripening periods, and that often have high BA concentrations, were screened for the presence of BA-degrading lactic acid bacteria (LAB). Seventeen isolates were found that were able to degrade tyramine and histamine in broth culture. All 17 isolates were identified by 16S rRNA sequencing as belonging to Lactobacillus casei. They were typed by plasmid S1-PFGE and genomic macrorestriction-PFGE analysis. Two strains (L. casei 4a and 5b) associated with high degradation rates for both BAs were selected to test how this ability might affect histamine and tyramine accumulation in a Cabrales-like mini-cheese manufacturing model. The quantification of BAs and the monitoring of the strains' growth over ripening were undertaken by RP-HPLC and qPCR respectively. Both strains were found to reduce histamine and tyramine accumulation. These two strains might be suitable for use as adjunct cultures for reducing the presence of BAs in cheese.     

Proteolytic and other hydrolytic enzyme activities in non-starter lactic acid bacteria (NSLAB) isolated from cheddar cheese manufactured in the United Kingdom

The populations of non-starter lactic acid bacteria (NSLAB) in a selection of 15 good-quality UK-manufactured Cheddar cheeses that had been matured for 6-9 months ranged from 10⁵ to 10⁷ bacteria g⁻¹. Fifteen different species of lactic acid bacteria were identified using commercially-available identification systems. The species isolated most frequently were Lactobacillus paracasei subsp. paracasei and Lb. plantarum; 10 other species were isolated from two or more cheeses and three species were recovered from only a single cheese. There were marked differences in the NSLAB populations of the cheeses produced by different UK manufacturers, and differences were also apparent in the populations of two cheeses produced on different occasions at the same creamery. Forty-one isolates, selected to include all the species identified and the dominant strains present in cheeses produced at several different creameries, were screened for activities of 34 proteolytic, five glycoside hydrolase and five esterolytic enzymes. All the NSLAB possessed a wide range of hydrolytic enzymes and therefore had the potential to contribute at some stage to the development of cheese flavour during the maturation and ripening period. Inter-species and strain differences in enzyme profiles and levels of activity were apparent and were determinants for the non-random selection of NSLAB for use as adjunct cultures in subsequent cheese-making trials. The breakdown of diagnostic substrates was indicative of the presence of multiple proteinase, tripeptidase, dipeptidase (including prolinase- and prolidase-like), dipeptidyl peptidase, prolyl, proline, aspartyl, pyroglutamyl (pyrrolidone-carboxyl) and general aminopeptidase activities.     

Selection, application and monitoring of Lactobacillus paracasei strains as adjunct cultures in the production of Gouda-type cheeses

Raw milk cheeses have more intense flavours than cheeses made from pasteurized milk and harbour strains with potential adjunct properties. Two Lactobacillus paracasei strains, R-40926 and R-40937, were selected as potential adjunct cultures from a total of 734 isolates from good quality artisan raw milk Gouda-type cheeses on the basis of their prevalence in different cheese types and/or over several production batches, safety and technological parameters. Conventional culturing, isolation and identification and a combined PCR-DGGE approach using total cheese DNA extracts and DNA extracts obtained from culturable fractions were employed to monitor viability of the introduced adjuncts and their effect on the cheese microbiota. The control cheese made without adjuncts was dominated by members of the starter, i.e. Lactococcus lactis and Leuconostoc pseudomesenteroides. In the cheeses containing either R-40926 or R-40937, the respective adjuncts increased in number as ripening progressed indicating that both strains are well adapted to the cheese environment and can survive in a competitive environment in the presence of a commercial starter culture. Principal component analysis of cheese volatiles determined by steam distillation-extraction and gas chromatography-mass spectrometry could differentiate cheeses made with different concentrations of adjunct R-40926 from the control cheese, and these differences could be correlated to the proteolytic and lipolytic properties of this strain. Collectively, results from microbiological and metabolic analyses indicate that the screening procedure followed throughout this study was successful in delivering potential adjunct candidates to enrich or extend the flavour palette of artisan Gouda-type cheeses under more controlled conditions.