Non-starter Lactic Acid Bacteria Strains Research Articles

Properties of an acid-tolerant, persistent Cheddar cheese isolate, Lacticaseibacillus paracasei GCRL163.

The distinctive flavours in hard cheeses are attributed largely to the activity of nonstarter lactic acid bacteria (NSLAB) which dominate the cheese matrix during maturation after lactose is consumed. Understanding how different strains of NSLAB survive, compete, and scavenge available nutrients is fundamental to selecting strains as potential adjunct starters which may influence product traits. Three Lacticaseibacillus paracasei isolates which dominated at different stages over 63-week maturation periods of Australian Cheddar cheeses had the same molecular biotype. They shared many phenotypic traits, including salt tolerance, optimum growth temperature, growth on N-acetylglucosamine and N-acetylgalactosamine plus delayed growth on D-ribose, carbon sources likely present in cheese due to bacterial autolysis. However, strains 124 and 163 (later named GCRL163) survived longer at low pH and grew on D-tagatose and D-mannitol, differentiating this phenotype from strain 122. When cultured on growth-limiting lactose (0.2%, wt/vol) in the presence of high concentrations of L-leucine and other amino acids, GCRL163 produced, and subsequently consumed lactate, forming acetic and formic acids, and demonstrated temporal accumulation of intermediates in pyruvate metabolism in long-term cultures. Strain GCRL163 grew in Tween 80-tryptone broths, a trait not shared by all L. casei-group dairy isolates screened in this study. Including citrate in this medium stimulated growth of GCRL163 above citrate alone, suggesting cometabolism of citrate and Tween 80. Proteomic analysis of cytosolic proteins indicated that growth in Tween 80 produced a higher stress state and increased relative abundance of three cell envelope proteinases (CEPs) (including PrtP and Dumpy), amongst over 230 differentially expressed proteins.

Effect of two thermoresistant non-starter lactic acid bacteria strains on volatilome profile during Cheddar ripening simulation

Dairy farm management practices can modify milk microbiota and therefore modulate non-starter lactic acid bacteria (NSLAB) found in cheese. These NSLAB can cause organoleptic defects. This study aimed to investigate the impact of two potential NSLAB in Cheddar cheesemaking: Lactiplantibacillus plantarum RKG 2-212 a strain isolated both in corn silage and raw milk, and Lactobacillus delbrueckii RKG R10, a strain isolated after pasteurisation of milk from a farm using grass and legume silage, and corn silage. The whole genome of these two lactobacilli was first sequenced. Then, the thermoresistance was evaluated after treatment at 60 °C for 5 min and compared to reference strains. Both lactobacilli were highly thermoresistant compared to other three lactic acid bacteria which are Lactococcus lactis subsp. cremoris ATCC 19257 and SK11, and L. plantarum ATCC 14917 (P < 0.0001). They lost less than 1 log cfu/mL (Δlog) and their genome contained a great number of copy number of genes coding for heat shock protein. During a Pearce test activity simulating Cheddar cheesemaking, the two lactobacilli did not show interaction with the starter Lcc. lactis subsp. cremoris SK11, and their population remained stable. During a ripening simulation, L. delbrueckii RKG R10 had a slight loss in viability in cheese slurry samples incubated at 30 °C for 12 d. However, L. plantarum RKG 2–212 had considerable growth, from 6.51 to 8.3 log cfu/g. This growth was associated with the acidification of the slurries (P < 0.0001). The presence of the lactobacilli modified the profile of volatile compounds evaluated by gas chromatography–mass spectrometry, accounting for 10.7% of the variation. The strain L. plantarum RKG 2-212 produced volatile compounds in greater quantity that could be associated with organoleptic defects such as acetic acid and 2-methylbutyraldehyde. Therefore, silage can be a vector of thermoresistant lactic acid bacteria for milk which can lead to flavor defects in cheese.

The potential of non-starter lactic acid bacteria from Cheddar cheese to colonise the gut

• NSLAB populations in Cheddar cheese can survive simulated gastric transit. • Many NSLAB strains capable of gastric transit exhibit probiotic traits. • Many strains bound to mucin suggesting potential for prolonged gut residence time. • No genes for antibiotic resistance, virulence or pathogenicity were identified. This study was undertaken to assess the potential of Non-Starter Lactic Acid Bacteria (NSLAB) from Cheddar cheese to survive gastric transit and display probiotic-related traits including bile salt hydrolase activity, the ability to adhere to the gut epithelium and inhibition of enteropathogen binding. Populations of NSLAB, up to 10 7 CFU/g per cheese were recovered following exposure of cheese to Simulated Stomach Duodenum Passage (SSDP) conditions. A total of 240 isolates were randomly selected from twelve Cheddar cheeses and assessed for probiotic traits. Two strains Lactobacillus paracasei DPC 7150 and Lactobacillus rhamnosus DPC 7102 showed the most probiotic potential. The Lb. paracasei and Lb. rhamnosus strains displayed adhesion rates of 64% and 79%, respectively and inhibited binding of pathogenic Escherichia coli by >20%. This research demonstrates that Cheddar cheese harbours potentially beneficial bacteria, a large portion of which can survive simulated digestion and potentially exhibit health beneficial effects once ingested.

Arthrospira platensis Extract: A Non-Invasive Strategy to Obtain Adjunct Attenuated Cultures.

This study aims at proposing the use of Arthrospira platensis, commonly known as Spirulina, extract as a non-invasive method to attenuate the growth rate of non-starter adjunct cultures, thus preventing the over-acidification that may occur during cheese manufacturing. A preliminary screening using four different concentrations (0.20%, 0.30%, 0.50%, and 0.70%) of A. platensis extract and four starter and three non-starter lactic acid bacteria strains was performed by impedometric analysis. This allowed us to select one starter and one non-starter strain to be used in the in vitro simulation of a co-culture in milk with the best antimicrobial concentration (0.3%). The growth dynamics of the two selected strains, starter Lactococcus lactis 1426 and non-sarter Lacticaseibacillus rhamnosus 1473, co-cultured for 120 h was monitored by three different approaches: (i) plate counting on M17, for the enumeration of lactococci, and MRS for lactobacilli; (ii) fluorescence microscopic counting of viable and non-viable coccoid Lactococcus lactis 1426 and rod-shaped Lacticaseibacillus rhamnosus 1473 cells; (iii) the overall estimation of co-culture growth behavior by impedometric parameters Lag, Rate, and yEnd. All the data obtained from the in vitro simulation were in agreement, revealing that a slowdown of non-starter growth occurred, while the starter strain was not affected, or slightly stimulated, from the antimicrobial presence. In particular, the growth of Lb. rhamnosus 1473 was delayed without adversely compromise the cells’ integrity, connected with metabolic functions, showing a great potential for use in cheese production.

Adjunct Culture of Non-Starter Lactic Acid Bacteria for the Production of Provola Dei Nebrodi PDO Cheese: In Vitro Screening and Pilot-Scale Cheese-Making.

The present study aimed at selecting non-starter lactic acid bacteria strains, with desirable technological and enzymatic activities, suitable as adjunct culture for the Provola dei Nebrodi cheese production. One hundred and twenty-one lactic acid bacteria, isolated from traditional Provola dei Nebrodi cheese samples, were genetically identified by Rep-PCR genomic fingerprinting, using the (GTG)5-primer, and by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-seven strains, included in the qualified presumption of safety (QPS) list, were tested for technological and proteinase/peptidase activities. Results showed that technological features and flavour formation abilities were strain-dependent. Among the selected strains, Lacticaseibacillus paracasei PN 76 and Limosilactobacillus fermentum PN 101 were used as adjunct culture in pilot-scale cheese-making trials. Data revealed that adjunct cultures positively affected the flavour development of cheese, starting from 30 days of ripening, contributing to the formation of key flavour compounds. The volatile organic compound profiles of experimental cheeses was significantly different from those generated in the controls, suggesting that the selected adjunct strains were able to accelerate the flavour development, contributing to a unique profile of Provola dei Nebrodi cheese.

Biogenic amine production by nonstarter strains of Lactobacillus curvatus and Lactobacillus paracasei in the model system of Dutch-type cheese

The objective of the study was to assess the development of the biogenic amine content in model cheese samples individually inoculated with two biogenic amine producing non-starter lactic acid bacteria strains of Lactobacillus curvatus subsp. curvatus (DEPE T3 and DEPE T36) and two biogenic amine producing non-starter lactic acid bacteria strains of Lactobacillus paracasei (DEPE T51 and DEPE T52) over the course of a 90 day storage period (10 ± 1 °C). During the entire ripening period, the dominant biogenic amine was tyramine. After just two months of ripening, tyramine concentration 167 mg/kg was detected in the model cheeses with the DEPE T3 strain (moreover a total biogenic amine concentration was 220 mg/kg), whilst in the case of the DEPE T36 strain it was even 211 mg/kg of tyramine (sum of biogenic amine was 302 mg/kg). In the samples with the added DEPE T51 and DEPE T52 strains, tyramine concentration after 90 days of ripening almost reached the level of 50 mg/kg.

Application of non-starter lactic acid bacteria as biopreservative agents to control fungal spoilage of fresh cheese

The effects of six non-starter lactic acid bacteria (NSLAB) as biopreservative agents against mycotoxigenic moulds in fresh cheeses were investigated. The potential of NSLAB to inhibit the growth of contaminating fungi was evaluated using end point (antifungal overlay assay and mycelium inhibition growth) and descriptive (antifungal assay on cell-free cheese extract) methods. The application of all NSLAB except Lactobacillus plantarum KU13 as the sole starter culture of fresh cheese resulted in significant delay of mycelial growth of Aspergillus flavus and Aspergillus parasiticus on the cheese surface. Compared with other NSLAB strains, Lb. plantarum PIN exhibited the most antifungal activity and delayed the mycelia growth of A. flavus and A. parasiticus on the cheese surface by 19 and 22 days, respectively (at 4 °C). The present study indicated that NSLAB could be used as a natural biopreservatives, for controlling the problems caused by mould growth in cheese.

Tailoring the probiotic potential of non-starter Lactobacillus strains from ripened Parmigiano Reggiano cheese by in vitro screening and principal component analysis

Non-starter lactic acid bacteria (NSLAB) inhabiting fermented food have been recently revised as source of probiotic strains. Here, we in vitro assessed the potential probiotic aptitude of a de-replicated set of NSLAB previously isolated from long ripened Parmigiano Reggiano cheeses (22 Lactobacillus rhamnosus, 18 Lactobacillus paracasei, 3 Lactobacillus casei, 2 Lactobacillus harbinensis, and 2 Lactobacillus fermentum). Most strains showed moderate to good resistance to biological barriers, including bile salts, lysozyme, and simulated gastric and pancreatic juices. Antimicrobial susceptibility tests against seven antimicrobials belonging to different categories showed that most strains are susceptible towards all the antibiotics, with the exception of vancomycin and streptomycin. The strains lost the streptomycin resistance when assayed on agar medium containing 0.5% bile salts, suggesting that detergent-like properties of cholic acids increase membrane permeability and mediate streptomycin susceptibility. No isolate showed bile salt hydrolase (BSH) activities, supporting that bile salts resistance and BSH activity are unpaired traits. Finally, NSLAB strains had moderate to high auto-aggregative and hydrophobic phenotypes, whereas two subsets of 22 and 8 strains co-aggregated with Escherichia coli and Salmonella enterica s. typhimurium, respectively. A multivariate analysis was effective to segregate one L. casei and two L. rhamnosus strains showing physiological characteristics compatible with probiotic properties.

Extension of Tosèla cheese shelf-life using non-starter lactic acid bacteria

Six strains of non-starter lactic acid bacteria (NSLAB) were used to extend the shelf-life of the fresh cheese Tosèla manufactured with pasteurised cows’ milk. The acidification kinetics of three Lactobacillus paracasei, one Lactobacillus rhamnosus and two Streptococcus macedonicus were studied in synthetic milk medium. Lb. paracasei NdP78 and NdP88 and S. macedonicus NdP1 and PB14-1 showed an interesting acidifying capacity and were further characterised for growth in UHT milk and production of antimicrobial compounds. Lb. paracasei NdP78 and S. macedonicus NdP1 grew more than 2 log cycles in 6 h. Lb. paracasei NdP78 was also found to produce a bacteriocin-like inhibitory substance (BLIS) active against Listeria monocytogenes. The four NSLAB strains (singly or in combination) were used to produce experimental pilot-scale cheeses which were compared by a panel. The cheese manufactured with the mixed culture Lb. paracasei NdP78 - S. macedonicus NdP1 was the most appreciated for its sensory properties. The cheeses produced at factory-scale showed higher concentrations of lactobacilli (7.90 log CFU/g) and streptococci (6.10 log CFU/g), but a lower development of coliforms (3.10 log CFU/g) and staphylococci (2.78 log CFU/g) than control cheese (4.86, 4.89, 4.93 and 5.00 log CFU/g of lactobacilli, streptococci, coliforms and staphylococci, respectively) processed without NSLAB addition. The food pathogens Salmonella spp. and Listeria monocytogenes were never detected. The dominance of the species inoculated was demonstrated by denaturing gradient gel electrophoresis (DGGE), whereas strain recognition was evaluated by randomly amplified polymorphic DNA (RAPD)-PCR. From the results obtained, Lb. paracasei NdP78 and S. macedonicus NdP1 were able to persist during the storage of Tosèla cheese and their combination influenced positively the sensory characteristics and shelf-life of the final product.

Non starter lactic acid bacteria biofilms: A means to control the growth of Listeria monocytogenes in soft cheese

The possibility to consider non starter lactic acid bacteria (NSLAB) biofilms as a means to control the growth of Listeria monocytogenes in soft cheeses was evaluated. In particular, the aptitude to form biofilm of four NSLAB strains ( Lactobacillus plantarum DSM1055, Lactobacillus casei DSM20011, Lactobacillus curvatus DSM20019 and Lactobacillus paracasei DSM20207) was investigated. All tested strains were able to form biofilm on stainless steel and the highest quantities were produced when NSLAB were present simultaneously (about 6 Log CFU cm −2). Then, experimental cheeses were made in presence of chips containing 7-days NSLAB biofilms and inoculated with L. monocytogenes (about 2 Log CFU g −1). Results demonstrated that NSLAB biofilms can be considered a useful means to delay the growth of L. monocytogenes in soft cheeses: the maximum cell load attained at the stationary phase was about 6 Log CFU g −1 in experimental cheeses against about 7 Log CFU g −1 observed in control samples.

Role of arginine in the development of secondary microflora in Swiss-type cheese

Abstract Microbial growth, lactose fermentation and casein breakdown in Swiss-type cheese was studied in 48 industrial trials. During cheese ripening, the average number of non-starter lactic acid bacteria (NSLAB) increased from 2.2×10 2 up to 1.4×10 7 cfu g −1 . The growth of bacteria was accompanied by the decrease of arginine concentration and formation of ornithine, NH 3 and citrulline. The average ornithine concentration in 3-month-old cheeses was 7.8±1.4 μmol g −1 . As ornithine is not a constituent of casein and some strains of NSLAB are able to convert arginine to ornithine, it was supposed that arginine could be one of the main energy sources for NSLAB in ripening cheese. According to our calculations, ATP available from arginine conversion to ornithine was theoretically sufficient to support the growth of up to 10 8 cfu NSLAB g −1 . This number higher than the maximum NSLAB numbers found in cheese suggested that sufficient amount of arginine was available in cheese. The experimental cfu numbers of NSLAB remained lower obviously due to the competition for available arginine with starter bacteria and the local unavailability of substrates and water. The pH-auxostat studies with the pure culture of Lactobacillus paracasei showed that the growth yield of lactobacilli was not significantly affected at typical cheese ripening temperatures T =7–12°C compared to optimal temperatures.

Biofilm Formation and Contamination of Cheese by Nonstarter Lactic Acid Bacteria in The Dairy Environment

Defects in cheese, such as undesirable flavors, gas formation, or white surface haze from calcium lactate crystals, can result from growth of nonstarter lactic acid bacteria (NSLAB). The potential for biofilm formation by NSLAB during cheese manufacturing, the effect of cleaning and sanitizing on the biofilm, and bacterial growth and formation of defects during ripening of the contaminated cheese were studied. Stirred-curd Cheddar cheese was made in the presence of stainless steel chips containing biofilms of either of two strains of erythromycin-resistant NSLAB (Lactobacillus curvatus strain JBL2126 or Lactobacillus fermentum strain AWL4001). During ripening, the cheese was assayed for total lactic acid bacteria, numbers of NSLAB, and percentage of lactic acid isomers. Biofilms of L. curvatus formed during cheese making survived the cleaning process and persisted in a subsequent batch of cheese. The starter culture also survived the cleaning process. Additionally, L. curvatus biofilms present in the vat dislodged, grew to high numbers, and caused a calcium lactate white haze defect in cheese during ripening. On the other hand, biofilms of L. fermentum sloughed off during cheese making but could not compete with other NSLAB present in cheese during ripening.Pulsed-field gel electrophoresis results verified the presence of the two biofilm strains during cheese making and in the ripening cheese. Probable contamination sites in the plant for other NSLAB isolated in the cheese were identified, thus supporting the hypothesis that resident NSLAB biofilms are a viable source of contamination in the dairy environment.

The ecology of non-starter lactic acid bacteria (NSLAB) and their use as adjuncts in New Zealand Cheddar

This paper reviews the recent New Zealand studies on non-starter lactic acid bacteria (NSLAB) in Cheddar cheese. In New Zealand, cheese adventitious NSLAB were usually strains of homofermentative lactobacilli and occasionally pediococci and heterofermentative lactobacilli. In Cheddar cheese, manufactured in six factories at different times and ripened over 24 months, over 140 NSLAB strains were identified using pulse field gel electrophoresis profiles and fermentation patterns on 22 carbohydrates. The majority of NSLAB isolates identified belong to two species: Lactobacillus paracasei subsp. paracasei and Lactobacillus rhamnosus. The composition of the NSLAB strains in a cheese was variable and dependent on the manufacturing factory, the date of manufacture and the age of the cheese. In most cheeses, no single NSLAB strain predominated during ripening. Up to six strains formed the majority (about 90%) of the NSLAB population, the remainder was represented by up to 15 other strains. In only about 10% of the cheeses investigated was one strain predominant throughout the 24 month ripening period. The acid and salt tolerance, effect of temperature, ability to produce biogenic amines and the lipolytic and proteolytic activities of the NSLAB strains were studied to screen strains for use as adjuncts. Sixty suitable strains were selected for further screening in a cheese model system. The NSLAB were incubated at 30°C for 10 days in the model system and the flavour and biochemical changes were assessed. Twenty-three strains were eliminated as potential adjuncts because they produced defective flavours and a further 13 strains were eliminated on the basis of undesirable biochemical properties (e.g. excess fermentation of glutamate, lactate racemisation). The 24 remaining NSLAB strains that had acceptable microbiological biochemical and flavour properties were used as adjuncts in Cheddar trials. Seven of the 24 adjunct combinations studied (made up from 2–4 strains of NSLAB) provided consistent flavour benefits. Analysis of the cheeses during ripening in these trials provided significant understanding of the ripening mechanisms of the NSLAB adjuncts.

Spatial and temporal distribution of non-starter lactic acid bacteria in Cheddar cheese.

The aim of this work was to investigate the spatial and temporal distribution of species and strains of non-starter lactic acid bacteria (NSLAB) within Cheddar cheese. Randomly amplified polymorphic DNA was used to identify and track the principle species and strain groups of NSLAB present. The same strains dominated each location examined within a cheese at any particular time point. Temporal change in species and strains of NSLAB during ripening was observed. A mixture of Lactobacillus paracasei, Lact. plantarum, Lact. rhamnosus and unidentified strains was found up to 6 weeks of maturation, thereafter only Lact. paracasei strains were isolated. Little variation in the spatial distribution of NSLAB strains occurs within Cheddar cheese; however, temporal changes in the species and strains were observed during ripening. The complex changes in the composition of the NSLAB community of Cheddar cheese may be the source of the variation in flavour that is seen in commercial practice.