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.     

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.     

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.     

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.     

A factory-scale application of secondary adjunct cultures selected from lactic acid bacteria during Puzzone di Moena cheese ripening

The lactic acid populations of 2 seasonal Puzzone di Moena cheeses made from winter and summer raw cow's milk were characterized at different ripening times. Lactic acid bacteria (LAB) were isolated on selective media and subjected to genetic typing and identification. The species most frequently found during ripening were Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, and Pediococcus pentosaceus. The different strains recognized by random amplification of polymorphic DNA-PCR were characterized for their acidifying and proteolytic activities to select nonstarter LAB to be used as secondary adjunct cultures (SAC). For each of the 3 above species, a strain showing weak acidification and high proteolytic capacity was selected. The 3 strains (Lb. paracasei ssp. paracasei P397, Lb. plantarum P399, and P. pentosaceus P41) constituted a mixed SAC used at 2 levels of concentration (10³ and 10⁴ cfu/mL) in experimental cheese making at dairy factory-scale. The analysis of volatile organic compounds as well as sensory analyses showed that the preferred level of SAC inoculation was 10³ cfu/mL.     

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.     

Dynamics of starter,adjunct non-starter lactic acid bacteria and propionic acid bacteria in low-fat and full-fat Dutch-type cheese

The microbial dynamics of Dutch-type cheeses differing in starter (commercial DL starter or single strain of Lactococcus lactis ssp. cremoris), adjunct (Lactobacillus or Propionibacterium) and fat contents (10% or 28% fat) were investigated by culture-dependent and culture-independent analysis. The cheese microbiota was dominated by the adjunct Lactobacillus after 4 weeks of ripening and the fat content did not influence the microbial diversity. The Leuconostoc sp., presumably from the DL starter, was detected in cheeses made with added Lactobacillus plantarum and Lactobacillus rhamnosus and was not detected in cheese made with added Lactobacillus paracasei after 4 and 7 weeks. No Lactobacillus spp. were detected in cheese with added Propionibacterium, while Leuconostoc was the only species detected. In cheeses made with Lc. lactis ssp. cremoris as starter, the Lactobacillus microbiota was similar to the cheese milk microbiota after 24 h while after 4 weeks different species of Lactobacillus and Leuconostoc were detected.     

Preliminary observations on proteolysis in Manchego cheese made with a defined-strain starter culture and adjunct starter (Lactobacillus plantarum) or a commercial starter

Different authors have demonstrated the potential of adding lactobacilli as adjunct cultures to pasteurized milk used in cheese manufacture. The aim of this work was to observe the effect of the use of a defined-strain starter culture and the addition of an adjunct culture (Lactobacillus plantarum) to cheesemilk in order to determine their effect on the ripening of Manchego cheese. Manchego cheeses were manufactured using one of the following starter culture systems: a defined starter consisting of Lactococcus lactis ssp. lactis and Leuconostoc mesenteroides ssp. dextranicum; a defined starter, as described above, and Lb. plantarum, which were isolated from a good quality Manchego cheese made from raw milk, or a commercial starter comprised of two strains of Lc. lactis. The cheeses were sampled at 15, 45, 90 and 150 d of ripening. Principal component analysis of peak heights of reversed-phase HPLC chromatograms of 70% (v/v) ethanol-insoluble and -soluble fractions distributed the samples according to the starter used in their manufacture. Quantitative differences in several peptides were evident between the three cheeses. Cheeses made with the defined-strain starters received higher scores for the flavour quality and intensity and for overall impression than the cheeses made with the commercial starter.     

Utilization of freeze-shocked lactobacilli for enhancing flavour development of Ras cheese

Freeze-shocked cultures of Lactobacillus helveticus or Lactobacillus casei were added at levels of 1% and 2% to Ras cheese milk prior to renneting as an adjunct starter to enhance flavour development of cheese. These additives did not affect the gross chemical composition of the cheeses but increased the formation of soluble nitrogenous compounds, free volatile fatty acids, the flavour intensity and improved the body characteristic. Also, the counts of bacterial groups (total, proteolytic and lipolytic) of the cheese treated with freeze-shocked lactobacilli were higher than in the control. Moreover, the ripening period was reduced to be 2 months compared with 4 months required for the control cheese. Also, using freeze-shocked culture of L. helveticus was the most effective in this respect.     

Influence of bacteria used as adjunct culture and sunflower oil addition on conjugated linoleic acid content in buffalo cheese

The influence of bacteria and sunflower oil addition on conjugated linoleic acid content (CLA) in buffalo cheese was determined. Fresh and short-ripened cheeses were manufactured using the same starter culture and four different adjunct strains previously selected by their CLA production rate. Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum and Streptococcus thermophilus were individually used as adjunct culture. Sunflower oil (SO) was added to obtain a final concentration of 200 μg/ml of linoleic acid. CLA levels in cheese were higher than raw milk, especially after ripening time. SO supplementation increase CLA concentrations in fresh cheeses, except in those manufactured with S. thermophilus as adjunct culture. Both, ripening and SO supplementation showed a positive influence on CLA concentration. Similar texture, acidity and colour were determined in cheeses with or without SO supplementation. Buffalo cheeses manufactured with appropriate adjunct cultures may be a natural source of CLA for human consumption.     

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.     

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.     

Potential of anticlostridial Lactobacillus isolated from cheese to prevent blowing defects in semihard cheese

Five anticlostridial Lactobacillus strains isolated from cheese were selected for a mixed adjunct culture. Cheese with the mixed adjunct culture (experimental) and without (control) was made in triplicate and ripened as vacuum‐packed and surface‐ripened cheese. Cheese gross composition was similar. Excessive gas formation occurred only in control cheeses. In contrast to control cheeses, the experimental cheeses were dominated by the added adjunct Lactobacillus strains (repetitive‐PCR). Casein breakdown was not influenced, however, the total amount of amino acids and pH was slightly lower in the experimental cheeses. Anticlostridial nonstarter Lactobacillus strains have potential as protective adjunct cultures against blowing defects in cheese.     

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.     

Anticlostridial activity of Lactobacillus isolated from semi-hard cheeses

Non-starter lactic acid bacteria grow to high numbers in semi-hard cheeses during ripening, and may suppress harmful bacteria. In this study, about 400 Lactobacillus isolates from Danish semi-hard cheeses were identified to species level using internal transcribed spacer-polymerase chain reaction (ITS-PCR) analysis. The majority of isolates belonged to the Lb. paracasei complex and were classified into approximately 135 types using pulsed field gel electrophoresis (PFGE). Lactobacillus isolates representing all the different PFGE types were screened, using an agar well diffusion assay, for antimicrobial activity against 15 single-strain Clostridium cultures. Almost half of the isolates possessed anticlostridial activity, and 10% possessed a broad and consistent activity. Nine strains were further investigated for properties of importance for use as mixed cultures in cheese and silage. The results showed that anticlostridial non-starter Lactobacillus growing in high quality semi-hard cheeses could be useful as protective adjunct cultures against the growth of Clostridium.     

Lactobacillus demonstrate thiol-independent metabolism of methylglyoxal: Implications toward browning prevention in Parmesan cheese

Endogenous production of α-dicarbonyls by lactic acid bacteria can influence the quality and consistency of fermented foods and beverages. Methylglyoxal (MG) in Parmesan cheese can contribute toward undesired browning during low temperature ripening and storage conditions, leading to the economic depreciation of affected cheeses. We demonstrate the effects of exogenously added MG on browning and volatile formation using a Parmesan cheese extract (PCE). To determine the influence of Lactobacillus on α-dicarbonyls, strains were screened for their ability to modulate concentrations of MG, glyoxal, and diacetyl in PCE. It was found that a major metabolic pathway of MG in Lactobacillus is a thiol-independent reduction, whereby MG is partially or fully reduced to acetol and 1,2-propanediol, respectively. The majority of lactobacilli grown in PCE accumulated the intermediate acetol, whereas Lactobacillus brevis 367 formed exclusively 1,2-propanediol and Lactobacillus fermentum 14931 formed both metabolites. In addition, we determined the inherent tolerance to bacteriostatic concentrations of MG among lactobacilli grown in rich media. It was found that L. brevis 367 reduces MG exclusively to 1,2-propanediol, which correlates to both its ability to significantly decrease MG concentrations in PCE, as well as its significantly higher tolerance to MG, in comparison to other lactobacilli screened. These findings have broader implications toward lactobacilli as a viable solution for reducing MG-mediated browning of Parmesan cheese.     

Evaluation of isolated starter lactic acid bacteria in Ras cheese ripening and flavour development

Eighteen cultures of starter lactic acid bacteria with or without added adjunct cultures, isolated from Egyptian dairy products, were evaluated in experimental Ras cheese for flavour development. Chemical composition of experimental cheeses was within the legal limit for Ras cheese in Egypt. All cultures used in this study had no effect on chemical composition of Ras cheese. Very significant variations in free amino acids, free fatty acids and sensory evaluation have been found among the cultures used in Ras cheesemaking. The levels of free amino acids and free fatty acids were correlated well with flavour development in Ras cheese. Seven of the tested cultures produced acceptable flavour and texture of Ras cheese. The highest overall score of flavour intensity, flavour and texture acceptability were in cheese made using YY47 lactic culture in addition to adjunct culture of Lactobacillus helveticus, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecium. This culture can be recommended for Ras cheese manufacture using pasteurized milk.     

Effect of addition of native cultures on characteristics of Armada cheese manufactured with pasteurized milk: A preliminary study

Six batches of Armada cheese were produced, one from raw milk with no added starter, another from pasteurized milk with a commercial mesophilic starter and four from pasteurized milk with experimental starters. These starters included: Lactococcus lactis subsp. lactis and L. lactis subsp. lactis biovar. diacetylactis; or the same strains plus either Enterococcus raffinosus, Leuconostoc mesenteroides subsp. dextranicum or Lactobacillus plantarum, all isolated from Armada cheese made from raw milk. The highest counts of aerobic mesophilic bacteria and populations of lactic acid bacteria for all batches were reached in the first week of ripening. These counts declined later throughout the ripening, although not at identical rates in every batch. Counts of lactobacilli were significantly higher in cheeses made from raw milk and those inoculated with the lactobacilli strain than in the other batches, over the whole ripening period. Added native starters minimized growth of Enterobacteriaceae. Cheeses made with the starter containing the Enterococcus strain had the most favourable sensory attributes throughout ripening.     

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.