Lactobacillus strains isolated from Danbo cheese as adjunct cultures in a cheese model system

Isolates of Non-Starter Lactic Acid Bacteria (NSLAB) from six ripened Danbo cheeses of different ages and of different brands were examined. Special emphasis was on the genus Lactobacillus with the aim of investigating their role in cheese maturation. Thirty-three isolates were typed by the PCR-based method, Randomly Amplified Polymorphic DNA (RAPD). Ten RAPD types were found and 70% of the isolates were of RAPD types found in more than one cheese. The different RAPD types were identified to species level by Temporal Temperature Gradient Gel Electrophoresis (TTGE). Most of the isolates were identified as Lactobacillus paracasei (76%), but also Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus rhamnosus and some taxa originating from the starter culture were detected. In one cheese, no lactobacilli were found.One strain of the most frequent Lactobacillus RAPD type from each of the five cheeses with a Lactobacillus flora was used as adjunct cultures in a cheese model system. Four of the five adjuncts were re-isolated during ripening. Two adjunct containing model cheeses received higher flavour scores than the control while two other were associated with off-flavours. The two model cheeses with off-flavour had a similar microflora and both were after 13 weeks of ripening dominated by a strain identified as L. plantarum.     

Proteolytic activity of three probiotic strains in semi-hard cheese as single and mixed cultures: Lactobacillus acidophilus,Lactobacillus paracasei and Bifidobacterium lactis

The influence of three probiotic strains (Lactobacillus acidophilus, Lactobacillus paracasei and Bifidobacterium lactis) in semi-hard cheese proteolysis patterns was assessed. Probiotics were inoculated both as single cultures and as a three-strain mix, and added to milk either after a pre-incubation step or directly to the vat. B. lactis did not show any effect on proteolysis of cheeses, while L. paracasei showed limited impact at the end of the ripening. In contrast, L. acidophilus significantly influenced secondary proteolysis from the beginning of ripening, causing an increase in the levels of small nitrogen-containing compounds and free amino acids and changes in the peptide profiles. The effect of Lactobacillus acidophilus on peptidolysis was more noticeable when it was added to cheese–milk after pre-incubation in an enriched milk fat substrate. Similar results obtained with the three-strain mixed culture, suggesting that L. acidophilus played a major role in secondary proteolysis of probiotic cheeses in this trial.     

Nonstarter Lactobacillus strains as adjunct cultures for cheese making: in vitro characterization and performance in two model cheeses

Nonstarter lactic acid bacteria are the main uncontrolled factor in today's industrial cheese making and may be the cause of quality inconsistencies and defects in cheeses. In this context, adjunct cultures of selected lactobacilli from nonstarter lactic acid bacteria origin appear as the best alternative to indirectly control cheese biota. The objective of the present work was to study the technological properties of Lactobacillus strains isolated from cheese by in vitro and in situ assays. Milk acidification kinetics and proteolytic and acidifying activities were assessed, and peptide mapping of trichloroacetic acid 8% soluble fraction of milk cultures was performed by liquid chromatography. In addition, the tolerance to salts (NaCl and KCl) and the phage-resistance were investigated. Four strains were selected for testing as adjunct cultures in cheese making experiments at pilot plant scale. In in vitro assays, most strains acidified milk slowly and showed weak to moderate proteolytic activity. Fast strains decreased milk pH to 4.5 in 8 h, and continued acidification to 3.5 in 12 h or more. This group consisted mostly of Lactobacillus plantarum and Lactobacillus rhamnosus strains. Approximately one-third of the slow strains, which comprised mainly Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus curvatus, were capable to grow when milk was supplemented with glucose and casein hydrolysate. Peptide maps were similar to those of lactic acid bacteria considered to have a moderate proteolytic activity. Most strains showed salt tolerance and resistance to specific phages. The Lactobacillus strains selected as adjunct cultures for cheese making experiments reached 10⁸ cfu/g in soft cheeses at 7 d of ripening, whereas they reached 10⁹ cfu/g in semihard cheeses after 15 d of ripening. In both cheese varieties, the adjunct culture population remained at high counts during all ripening, in some cases overcoming or equaling primary starter. Overall, proximate composition of cheeses with and without added lactobacilli did not differ; however, some of the tested strains continued acidifying during ripening, which was mainly noticed in soft cheeses and affected overall quality of the products. The lactobacilli strains with low acidifying activity showed appropriate technological characteristics for their use as adjunct cultures in soft and semihard cheeses.     

Genotypic and technological diversity of Leuconostoc mesenteroides and Lactobacillus paracasei subsp. paracasei strains for use as adjunct starter cultures in Pecorino di Filiano cheese

Seventeen Leuconostoc mesenteroides and 33 Lactobacillus paracasei subsp. paracasei from traditional Pecorino di Filiano cheese were tested for their potential use as adjunct starters, and a study of their genetic variability was carried out. Forty one per cent (41%) of Leu. mesenteroides and 46% of Lb. paracasei subsp. paracasei showed medium–high proteolytic activity, while high lipolytic activity was detected in 18% of Lb. paracasei subsp. paracasei. The aminopeptidase activity of Lb. paracasei subsp. paracasei was higher than that of Leu. mesenteroides. Strain diversity by RAPD analysis showed a high degree of heterogeneity. This study identified strains with unusual properties that could be good candidates as adjuncts in a starter to manufacture PF cheese.     

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.     

Use of wild Lactobacillus strains in an adjunct culture for a Roncal-type cheese

The effect of an added adjunct culture consisting of facultatively heterofermentative lactobacilli (FHL) on the volatile compounds and sensory characteristics of a Spanish ewes'-milk cheese was examined. Three cheese batches were prepared using a commercial starter, one from raw milk, another from pasteurized milk, and a third from pasteurized milk with an added culture of wild Lactobacillus. paracasei+Lb. plantarum. Analysis of the volatile compounds was carried out by the purge and trap method and gas chromatography with a mass spectrometer and disclosed a total of 86 compounds belonging to the chemical families hydrocarbons, fatty acids, esters, ketones, aldehydes, and alcohols. After ageing for 120 and 240 days, the cheese samples underwent sensory analysis by a panel of expert assessors. The attributes evaluated were characteristic odour and odour intensity and characteristic aroma and aroma intensity. Pasteurization of the milk had an effect on the formation of certain volatile compounds, adversely affecting the characteristic flavour of the cheese. Use of the adjunct culture in addition to the commercial starter improved the flavour of the cheese made from the pasteurized milk, which earned sensory scores similar to those awarded to the cheese made from the raw milk. Use of adjunct cultures consisting of indigenous FHL strains could help to conserve the traditional characteristics of Roncal cheese made from pasteurized milk, although some technical adjustments to the Regulations would be needed.     

Isolation and characterisation of exopolysaccharide-producing Weissella and Lactobacillus and their application as adjunct cultures in Cheddar cheese

This study characterised exopolysaccharide-producing lactic acid bacteria and examined their potential for use in Cheddar cheese manufacture. Two strains were chosen for incorporation as adjunct cultures in Cheddar cheese manufacture: namely, the homopolysaccharide-producers Weissella cibaria MG1 and Lactobacillus reuteri cc2. These strains both produce dextrans with molecular masses ranging from 10⁵ to 10⁷ Da. Both strains were used in the production of miniature Cheddar cheeses that employed a conventional commercial cheese starter culture Lactococcus lactis R604. A cheese was also included that used purified dextran as an ingredient. The W. cibaria strain survived in cheese with levels increasing by 1.5 log cycles over the ripening period. All experimental cheeses (adjunct or exopolysaccharide ingredient) had higher moisture levels compared with the control cheese made using starter alone. Inclusion of the adjunct strains had no detectable negative effects on cheeses in terms of proteolysis.     

Judge selection for hard and semi-hard cheese sensory evaluation

During a five month period, 34 potential panellists were subjected to 21 selection tests (triangular test, threshold evaluation, ranking test and stimulus recognition test) based on taste, aroma and textural characteristics of cheese. Standardised available ricotta cheese was used as a substrate in taste and aroma tests, while commercially available cheese was utilised to evaluate candidate performance in textural characteristic tests. A combination of procedures (binomial, sequential and bi-plot) allowed the definition of homogeneous subsets of candidates suitable for different analysis. The binomial approach allowed the last performing candidates to be identified while the sequential procedure enhanced our understanding of their skills. To make the selection criteria more coherent the performance parameters were objectively derived from the array of tests. Candidates were classified into three groups, depending on their capabilities, and the different discrimination power of the tests was highlighted through bi-plots.     

Autolysis of selected Lactobacillus helveticus adjunct strains during Cheddar cheese ripening

Cheddar cheeses were manufactured on a pilot scale (500 L vats) with three different Lactobacillus helveticus strains, which showed varying degrees of autolysis, added as adjuncts to the starter. Autolysis of adjunct strains was monitored by reduction in cell numbers, level of intracellular enzymes released into the cheese, and by the consequent changes in the degree of proteolysis and concentration of free amino acids in the cheese. The flavour profiles of the cheeses at 6 months were also determined. Significant variation in viability of the Lb. helveticus strains, which showed a positive correlation with the indicators of autolysis, was observed. However, cheese manufactured with the most autolytic strain did not receive the highest flavour scores. The results indicate that whereas autolysis of adjunct strains is an important factor in Cheddar cheese flavour development, other factors also contribute to the overall flavour improvement observed.     

Mould contamination in production of semi-hard cheese

Air, equipment, plastic film, brine and milk were sampled from four cheese factories in Norway during the period September 1997 to May 1999 in order to identify the critical points for mould contamination in the production process. Altogether, 672 samples were collected. Penicillium brevicompactum was the most frequently isolated species from three of the factories, while Geotrichum candidum was the most frequently isolated species from the fourth. P. commune, P. palitans, P. solitum and P. roqueforti ss. roqueforti, all common contaminants on cheese, were found in samples of air and equipment, and the former was also isolated from plastic film. The results in the present study showed that the mould levels in the cheese factories varied between the different control points. The mould levels at some of the air control points had high mould counts while the mould levels in milk and brine, on equipment and on plastic film, generally were low. The fungi at the control points with high mould levels consisted of common cheese contaminants as well as species not commonly isolated from cheese. The statistical analysis showed that air was the major source of the important cheese contaminants P. commune and P. palitans during the production process. High quality air with low number of cheese contaminants in production rooms, especially the wrapping room, is important in order to reduce mould contamination.     

Aroma development in semi-hard reduced-fat cheese inoculated with Lactobacillus paracasei strains with different aminotransferase profiles

Reduced-fat, semi-hard round-eyed cheese was made from bovine milk with a mesophilic DL-starter and three Lactobacillus paracasei subsp. paracasei single-strain adjuncts with different aminotransferase (AT) activity profiles. The pilot-plant procedure was not influenced by the adjuncts, and similar cheese was made from all vats. The growth of each adjunct to dominate the cheese was confirmed by using DNA fingerprinting. Flavour profiles were different for cheeses made with the different Lactobacillus strains. Use of the adjunct CHCC 4256 significantly increased the content of flavour compounds that were produced from the branched-chain amino acids (BcAAs: Leu, Ile and Val) and Asp. These cheeses also had superior sensory characteristics as they tasted aromatic and sweet without bitterness. The adjunct CHCC 4256 did not have glutamate dehydrogenase activity, or the highest activity on BcAA, but showed typical AT activity, with a similar activity on Leu, Phe and Asp substrates.     

Behaviour of Escherichia coli strains during semi-hard and hard raw milk cheese production

The behaviour of two Escherichia coli strains, one with a high and the other with a low thermotolerance phenotype, was investigated during production and ripening of Swiss semi-hard and hard raw milk cheese. In semi-hard cheese, counts of E. coli increased during production, before a log-linear decrease occurred during ripening, with a faster rate of reduction in core than in rind samples, and faster reduction of the more heat-sensitive strain in rind samples. Nevertheless, at the end of semi-hard cheese ripening, E. coli were present at least at 1.3 log₁₀ cfu g⁻¹ in rind samples and remained detectable by enrichment of core samples. During the first day of hard cheese production, both E. coli strains were almost completely inactivated. Detection by enrichment was possible in one of twelve spiked cheeses after 16 weeks, indicating the potential of a thermotolerant E. coli strain to survive until the end of ripening.     

Effect of wild strains used as starter cultures and adjunct cultures on the volatile compounds of the Pecorino Siciliano cheese

The effect of six wild strains on the volatile profile of the PS cheese was investigated and compared to that generated from industrial starters generally used to produce PS cheese. All cheeses were subjected to microbiological, physicochemical, and volatile compounds analyses. The DGGE of the 16S rDNA analysis was also applied. The volatile compounds generated during ripening were studied through the SPME and the GC-MS methods. No difference was detected between the experimental and control cheeses throughout chemical and microbiological analyses, while the DGGE results showed the presence of Streptococcus thermophilus in all cheeses, and the dominance of Enterococcus durans, Lactobacillus rhamnosus, and Lactobacillus casei in most of the experimental cheeses. Moreover, the presence of Lactococcus lactis species as in the control and in the experimental P2 and P4 cheeses was also revealed. The SPME results showed more pronounced volatile compounds in the experimental cheese samples than in the control ones.     

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.     

Biogenic amine content and proteolysis in Manchego cheese manufactured with Lactobacillus paracasei subsp. paracasei as adjunct and other autochthonous strains as starters

Two different autochthonous strain starter cultures, in which the acidifying starter was composed of strains of Lactococcus lactis, were used for the manufacture of pasteurised milk Manchego cheese. Proteolysis parameters, biogenic amines and sensory characteristics were evaluated and compared with those of commercial starter Manchego cheese and raw milk Manchego cheese manufactured without starter. Autochthonous starter cheeses, and especially those including Lactobacillus paracasei subsp. paracasei as adjunct, presented higher levels of proteolysis than in commercial starter cheese. The concentrations of total biogenic amines in autochthonous starter cheeses were much lower than in raw milk cheese and even lower than in commercial starter cheese. Cheese manufactured with the adjunct strain gave the best results for both flavour intensity and flavour quality, and was the most preferred by panellists. The results suggest that the culture containing Lb. paracasei subsp. paracasei as adjunct could be used for the manufacture of industrial Manchego cheese.     

Tyramine production in Dutch-type semi-hard cheese from two different producers

Tyramine content and counts of lactic acid bacteria (LAB) and enterococci were measured (including tyrosine-decarboxylase activity testing, and testing of the presence of the tyrosine-decarboxylase gene sequence, tyrdc, by PCR) during ripening (0, 26, 54, 88, 119, 146, and 176 days) in the core (C)- and edge (E)-samples of Dutch-type semi-hard cheese produced from pasteurized milk by two dairies (R, H) with two levels of fat content (30 and 45%) using two different starter cultures (Y, L), respectively. Tyramine content (y, mgkg(-1)) increased (P<0.001) with increasing time of ripening (x, days) in the cheeses of both producers (R: y=0.88x-31.4, R(2)=0.30; H: y=0.50x-6.3, R(2)=0.18), and its content was higher (P<0.01) in E-samples in comparison with C-samples. Time of ripening, part of the cheese and starter culture accounted for 67%, 28%, and 4% of explained variability of tyramine content in the cheese, respectively. After 26 days of ripening, using decarboxylase screening medium (DCM), tyrosine-decarboxylase positive LAB isolates constituted 7-27% and 6-32% of the square root of total countable colonies of LAB isolates of the producer R and H, respectively; tyrosine-decarboxylase positive enterococci were present only in R-cheeses (4-26% of the square root of total countable colonies). Tyrdc was confirmed only in 13% and 42% of the tyrosine-decarboxylase positive LAB and enterococci isolates, respectively (presumably due to the tendency of DCM to give false-positive results). Lactobacillus curvatus subsp. curvatus and Enterococcus durans, Enterococcus faecalis, and Enterococcus casseliflavus were identified as tyrdc-positive LAB and enterococci in the cheeses, respectively.     

Probiotic bacteria as adjunct starters: influence of the addition methodology on their survival in a semi-hard Argentinean cheese

Two strains of probiotic bacteria, one of Lactobacillus acidophilus and the other of Lactobacillus paracasei subsp. paracasei, were tested as adjunct cultures in cheese-making experiments, in order to assess their viability during cheese-making and ripening. The adjunct culture was added to cheese-making milk following two different methodologies: as a lyophilized powder dispersed in milk, or within a substrate composed of milk and milk fat. In all cheeses, probiotic bacteria increased a log cycle during cheese-making, and remained almost constant during ripening (60 days), always in higher number than required to meet probiotic standards. Gross composition of the cheeses was not affected by the addition of probiotic bacteria, except for pH value: cheeses with L. acidophilus added within the pre-incubated substrate, had lower pH values and were over acidified and crumbly. Direct addition of the probiotic culture was the methodology with the best performance; however the pre-incubation presented some advantages such as an increased population of lactobacilli in the initial inoculum.     

用于低脂干酪开发的附属发酵剂的筛选

目的筛选出生产低脂干酪所需添加较为适宜的附属发酵剂。方法选择7株乳酸菌作为研究对象,测定其总肽酶活力、菌株生产能力、产酸能力、产粘能力、菌体自溶度、产胞外多糖数量、蛋白质水解能力等7种指标。结果 7株乳酸菌中嗜酸乳杆菌(Lactobacillus acidophilus,LA)总肽酶活力最高,为(256.46±11.88)μg/L,植物乳杆菌(Lactobacillus plantarum,LP)最低;菌株生长能力LA最好;菌株副干酪乳杆菌(Lactobacillus paracasei,LPC)在发酵10 h后的产酸能力最强且趋于稳定,pH在3.8左右;LP菌株产粘能力最优,为(168.10±14.72)mPa·s;瑞士乳杆菌(Lactobacillus helveticus,LH)菌株自溶度最好,达到35%;产胞外多糖数量最优菌株是LP,含量为(792.69±35.94)mg/L;LH的蛋白质水解能力最高,水解产生的游离氨基酸含量为(94.78±2.82)mg/L。结论 LH作为低脂干酪的附属发酵剂较好。     

Heat resistance of Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk

Nine strains of non-starter Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk were selected for their anticlostridial activity. Resistance to thermisation (60 °C, 5 min) and pasteurisation (73 °C, 15 s) was investigated using a submerged-coil apparatus. MRS broth-grown cultures of all nine strains survived thermisation in buffer. The level of resistance to thermisation was strain dependent and lower for freshly grown cells (stationary phase cells) than for resting cells (freshly grown cells kept diluted 10-fold in MRS broth at 17 °C for 6 days). None of the nine Lb. paracasei strains survived or recovered after pasteurisation in buffer when grown in MRS broth, while seven of the nine strains survived pasteurisation in UHT whole milk when grown in milk. Identity of the strains was successfully confirmed during the experiments using repetitive-PCR analysis. The potential of Lb. paracasei strains to survive pasteurisation of cheese milk was demonstrated.     

Assessing the proteolytic and lipolytic activities of single strains of mesophilic lactobacilli as adjunct cultures using a Caciotta cheese model system

Seventeen strains of mesophilic lactic acid bacteria, isolated from cheese (non-starter lactic acid bacteria, NSLAB) or sourdough, were used individually as adjunct cultures in a Caciotta cheese model system. Adjunct cultures were monitored by randomly amplified polymorphic DNA analysis and their cell counts mainly varied from ca. 9.0 to 8.0 log cfu g⁻¹ throughout 36 days of ripening. Adjunct cultures influenced differently cheese proteolysis. Both NSLAB and sourdough strains caused an extensive secondary proteolysis; however, some NSLAB strains produced the highest concentration of free amino acids. Principal component analysis (PCA) differentiated cheeses manufactured with NSLAB strains Lactobacillus parabuckneri B₉F_ST, Lb. paracasei B₆₁F₅, Lb. curvatus 2768 and Lb. rhamnosus ATCC 7469 based on the accumulation of Lys, Glu, Phe, Hist, Asp and Met. Assessment of cheese lipolysis showed that: (i) highest concentrations of free fatty acids (FFA) were found with NSLAB strains Lb. rhamnosus ATCC 7469 and Lb. casei subsp. pseudoplantarum 2742 (ca. 10 500 mg kg⁻¹); (ii) PCA differentiated cheeses manufactured with NSLAB strains Lb. rhamnosus ATCC 7469 and Lb. casei subsp. pseudoplantarum 2742 based on the accumulation of palmitic (C16:0) and linoleic (C18:2) acids, and those with Lb. curvatus 2768 and Lb. parabuckneri B₉F_ST based on the high concentration of short chain FFA; (iii) the cheese made with sourdough strain Lb. sanfranciscensis CB1 had the highest levels of unsaturated FFA.