Lactococcal Strains Research Articles

Microbial interactions shape cheese flavour formation

Cheese fermentation and flavour formation are the result of complex biochemical reactions driven by the activity of multiple microorganisms. Here, we studied the roles of microbial interactions in flavour formation in a year-long Cheddar cheese making process, using a commercial starter culture containing Streptococcus thermophilus and Lactococcus strains. By using an experimental strategy whereby certain strains were left out from the starter culture, we show that S. thermophilus has a crucial role in boosting Lactococcus growth and shaping flavour compound profile. Controlled milk fermentations with systematic exclusion of single Lactococcus strains, combined with genomics, genome-scale metabolic modelling, and metatranscriptomics, indicated that S. thermophilus proteolytic activity relieves nitrogen limitation for Lactococcus and boosts de novo nucleotide biosynthesis. While S. thermophilus had large contribution to the flavour profile, Lactococcus cremoris also played a role by limiting diacetyl and acetoin formation, which otherwise results in an off-flavour when in excess. This off-flavour control could be attributed to the metabolic re-routing of citrate by L. cremoris from diacetyl and acetoin towards α-ketoglutarate. Further, closely related Lactococcus lactis strains exhibited different interaction patterns with S. thermophilus, highlighting the significance of strain specificity in cheese making. Our results highlight the crucial roles of competitive and cooperative microbial interactions in shaping cheese flavour profile.

Molecular mechanisms underlying the structural diversity of rhamnose-rich cell wall polysaccharides in lactococci

In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using invitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.

Сохранение жизнеспособности и свойств коллекционных культур молочнокислых бактерий при длительном хранении

The study featured the viability of lactic acid bacteria during long-term storage at 2-4 C. The stock cultures were preserved by vacuum drying with no freezing or lyophilization. In general, the lactococci demonstrated a high survival rate after 3-54 years of storage: the residual viable cell count was 0.98-43 million CFU per ampoule. However, 8.3 % out of 3,552 lactococcal strains demonstrated a significant decrease in acid-inducing activity. Thirty strains, which had been used to produce bacterial starters for the past 25 year, lost their viability. Only 12.9 % of the lactobacilli that had been sublimated 62 years ago could not be revived.

Antimicrobial Peptides (Bacteriocins) Produced by Lactococcus lactis and Pediococcus pentosaceus Strains with Activity Against Clinical and Food-Borne Pathogens.

Bacteriocins are ribosomal-synthesized peptides with antimicrobial activity, produced by different groups of bacteria, including lactic acid bacteria (LAB). Most of the produced by LAB bacteriocins can be described with rather broad spectra of inhibition and they offer suggested applications in food preservation and pharmaceutical sector. Different LAB were isolated from fermented food products and fruits, obtained from the region of Pohang, Korea, and identified based on physiological, biochemical, and molecular methods. The promising isolates, Pediococcus pentosaceus 732, Lactococcus lactis 808, and Lactococcus lactis subsp. lactis 431, were identified based on biochemical, physiological, and biomolecular approaches, including 16S rRNA partial sequencing, and were evaluated for production of bacteriocin, including stability in presence of enzymes, chemicals, pH, and temperatures. Adherence properties for the expressed bacteriocins by P. pentosaceus 732, Lc. lactis 808, and Lc. lactis subsp. lactis 431 were evaluated at presence of selected chemicals, pH, and temperatures. The presence of bacteriocin genes in the strains was investigated and analyzed. The bacterial effect of bacteriocin produced by studied strains on Listeria spp. and Staphylococcus spp. has been shown for actively growing and stationary cells. Similar growth and bacteriocin production were observed when studied strains were cultured in MRS at 30°C or 37°C. The presence of nisin operon with some point mutations on the genomic DNA was recorded based on the performed PCR reactions targeting different genes associated with nisin expression for both lactococcal strains. Pediocin PA-1 operon was evaluated in a similar manner for P. pentosaceus 732.

A Metagenomics Approach to Enumerate Bacteriophages in a Food Niche.

Dairy fermentation relies on the activity of starter cultures composed primarily of lactic acid bacteria such as Lactococcus and Streptococcus thermophilus strains to produce consistent, high-quality products. Bacteriophages are a constant threat to the industry, often causing slowed or failed fermentation resulting in significant economic losses. To ensure the continuation of reliable fermentation practices, it is important to detect and monitor the phage populations impacting different starter cultures. This has traditionally been done primarily through culture-dependent methods but has since expanded into viral metagenomics. Here we outline a protocol for a targeted virome extraction from a dairy whey sample, followed by subsequent sequencing and phageome analysis of the sample.

An in vitro gastrointestinal model to evaluate the tolerance of encapsulated Lactobacillus and Lactococcus strains with synbiotic containing lactobionic acid via lyophilization technique to harsh gastric conditions during storage time

Probiotics are living microorganisms that have potential health benefits for the host when administered in the correct proportions. However, for these beneficial effects to be produced, viable probiotic cells need to reach the human intestine. This study used a gastrointestinal model to represent the pH variation and gastric enzymes that are involved in the digestion process. Eight Lactobacillus strains and one Lactococcus strain were encapsulated using sodium alginate. The aim of this study was to compare free probiotic bacteria with encapsulated probiotic bacteria and determine the number of viable probiotic cells that might reach the human intestine. Microencapsulation of probiotic organisms followed the extrusion method with an alginate compound. Microencapsulated and free probiotic bacteria were inoculated into 200 ml of MRS (Man, Rogosa, Sharpe) broth. Simulated gastric juice and bile and pancreatic salt were used to test the tolerance of free Lactobacillus and Lactococcus species and microencapsulated bacteria to acid pH and enzymes. To enumerate the microencapsulated probiotic organisms, the bacteria were released from the capsules after total digestion with sodium citrate solution. The number of live bacteria (as CFU/g) was determined using the plate count method on MRS plates incubated at 37 °C for 48 h. Bacterial cell viability was determined on MRS agar duplicate plates at different dilutions. The results indicated that microencapsulation of probiotic bacteria showed improved viability over free cells when given 2 h of exposure to acidic conditions, and also for 2 h of exposure to bile and pancreatic salt. Encapsulation with alginate can protect probiotic bacteria from acidic conditions and bile salts. This study attempted also to produce synbiotic preparations with lactobionic acid using nontoxic encapsulating substances. The microorganisms used were stabilized with protective substances, and their stability during storage was checked under room temperature conditions. The final modification of the method, involving placing the capsules in a solution of skimmed milk with 5 % sucrose and 0.35 % ascorbic acid, resulted in an increase in the survival rate of some strains of microorganisms during microencapsulation and lyophilization and demonstrated stability during storage at room temperature.

Lactic acid bacteria from artisanal raw goat milk cheeses: technological properties and antimicrobial potential.

In cheese-making, a starter culture composed of adequately chosen lactic acid bacteria (LAB) may be suitable to ensure the rapid acidification of milk, improve textural and sensory characteristics, and avoid pathogen proliferation. In this work, 232 LAB isolates collected from artisanal goat's raw milk cheeses produced in Portugal were evaluated for their antimicrobial capacity (at 10 and 37°C), as well as their acidifying and proteolytic properties. Among the 232 isolates, at least 98% of those isolated in De Man- Rogosa-Sharpe (MRS) agar presented antagonism against Listeria monocytogenes, Salmonella Typhimurium, or Staphylococcus aureus, whereas less than 28.1% of M17-isolated LAB showed antagonism against these pathogens. M17-isolated LAB displayed better results than MRS ones in terms of acidifying capacity. As for the proteolytic assay, only 2 MRS isolates showed casein hydrolysis capacity. Principal component analyses and molecular characterization of a subset of selected isolates were conducted to identify those with promising capacities and to correlate the identified LAB genera and species with their antimicrobial, acidifying, and/or proteolytic properties. Lactococcus strains were associated with the highest acidifying capacity, whereas Leuconostoc and Lacticaseibacillus strains were more related to antimicrobial capacities. Leuconostoc mesenteroides, Lactococcus lactis, and Lacticaseibacillus paracasei were the predominant organisms found. The results of this work highlight various strains with pathogen inhibition capacity and suitable technological properties to be included in a customized starter culture. In future work, it is necessary to appropriately define the starter culture and implement it in the cheese-making process to evaluate if the in-vitro capacities are observable in a real food system.

Improvement of Locomotion Caused by Lactococcus lactis subsp. lactis in the Model Organism Caenorhabditis elegans.

Lactococcus lactis subsp. lactis exhibits probiotic properties in humans. Considering that Caenorhabditis elegans can be used to study the effects of microorganisms on animal behavior, owing to its simple nervous system, we assessed the impacts of two strains of Lactococcus lactis subsp. Lactis-a non-nisin-producing strain, NBRC 100933 (LL100933), and a nisin-producing strain, NBRC 12007 (LL12007)-on the lifespan, locomotion, reproductive capacity of, and lipid accumulation in, C. elegans. The lifespan of adult C. elegans fed a mixture (1:1) of Escherichia coli OP50 and LL100933 or LL12007 did not show a significant increase compared to that of the group fed a standard diet of E. coli OP50. However, the nematodes fed Lactococcus strains showed notable enhancement in their locomotion at all of the tested ages. Further, the beneficial effects of LL100933 and LL12007 were observed in the daf-16 mutants, but not in the skn-1 and pmk-1 mutants. The lipid accumulation in the worms of the Lactococcus-fed group was lower than that in the control group at all experimental ages. Overall, LL100933 and LL12007 enhance the locomotor behavior of C. elegans, likely by modulating the PMK-1/p38 MAPK and SKN-1/Nrf2 transcription factors.

Fermentative production of 2,3-butanediol from cheese whey by a non-engineered mutant strain of Lactococcus lactis

Cheese manufacturing generates huge amounts of whey, part of it being disposed of as waste, posing serious environmental problems. However, whey is a valuable resource that can be valorized in a cost-effective and sustainable way, being fermentation processes a suitable option. The non-engineered mutant strain Lactococcus lactis 43103, a non-pathogenic microorganism, was found to efficiently produce 2,3-butanediol (2,3-BDO) from lactose. Culture of this strain in a whey-based medium adapted to its nutritional requirements under optimized fermentation conditions resulted in 2,3-BDO productions close to 18 g/L, representing 76.8 % of the maximum theoretical yield, which reached 94.7 % when acetoin, its immediate precursor in the metabolic pathway, was included. The fermentation performance was not very far from that of the best producers (natural or engineered) and was the best one among the non-pathogenic and non-engineered bacteria. These results open up the possibility of valorizing whey through fermentation to 2,3-BDO, a valuable platform chemical.

Reduced synthesis of phospho-polysaccharide in Lactococcus as a strategy to evade phage infection

Lactococcus spp. are applied routinely in dairy fermentations and their consistent growth and associated acidification activity is critical to ensure the quality and safety of fermented dairy foods. Bacteriophages pose a significant threat to such fermentations and thus it is imperative to study how these bacteria may evade their viral predators in the relevant confined settings. Many lactococcal phages are known to specifically recognise and bind to cell wall polysaccharides (CWPSs) and particularly the phospho-polysaccharide (PSP) side chain component that is exposed on the host cell surface. In the present study, we generated derivatives of a lactococcal strain with reduced phage sensitivity to establish the mode of phage evasion. The resulting mutants were characterized using a combination of comparative genome analysis, microbiological and chemical analyses. Using these approaches, it was established that the phage-resistant derivatives incorporated mutations in genes within the cluster associated with CWPS biosynthesis resulting in growth and morphological defects that could revert when the selective pressure of phages was removed. Furthermore, the cell wall extracts of selected mutants revealed that the phage-resistant strains produced intact PSP but in significantly reduced amounts. The reduced availability of the PSP and the ability of lactococcal strains to revert rapidly to wild type growth and activity in the absence of phage pressure provides Lactococcus with the means to survive and evade phage attack.

Mechanisms underlying probiotic effects on neurotransmission and stress resilience in fish via transcriptomic profiling

In recent years, studies have highlighted the significant impact of probiotic treatment on the central nervous system (brain) and stress regulation through the microbiota-gut-brain axis, yet there have been limited knowledge on this axis in fish. Therefore, this study aimed to enhance the current understanding of the mechanisms underlying probiotic effects on neurotransmission and stress alleviation in fish through transcriptomic profiling. In this study, olive flounders (Paralichthys olivaceus) were subjected to two trial setups: a 1-month lab-scale trial and a 6-month field-scale trial, with and without the probiotic strain Lactococcus lactis WFLU12. RNA-Seq analysis was performed using liver samples collected from fish at one-month post-feeding (mpf) in both trials. Additionally, fish growth was monitored monthly, and serological parameters were measured at one mpf in the field-scale experiment. The results of the lab-scale trial showed that probiotic administration significantly upregulated genes related to neurotransmission, such as htr3a, mao, ddc, ntsr1, and gfra2. These findings highlight the impact of probiotics on modulating neurotransmission via the microbiota-gut-brain axis. In the field-scale experiment, fish growth was significantly promoted and the sera levels of AST, LDH, and cortisol were significantly higher in the control group compared to the probiotics group. Furthermore, genes involved in stress responses (e.g. hsp70, hsp90B1, hspE1, prdx1, and gss) and transcriptional regulators (e.g. fos, dusp1, and dusp2) exhibited significant upregulation in the control group compared to the probiotics group, indicating that probiotic administration can alleviate stress levels in fish. Overall, this study provides valuable insights into the mechanisms underlying the beneficial effects of probiotics in fish, specifically regarding their impact on neurotransmission and stress alleviation.

Microbiome of Zoophytophagous Biological Control Agent Nesidiocoris tenuis.

Many insects are associated with endosymbionts that influence the feeding, reproduction, and distribution of their hosts. Although the small green mirid, Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae), a zoophytophagous predator that feeds on plants as well as arthropods, is a globally important biological control agent, its microbiome has not been sufficiently studied. In the present study, we assessed the microbiome variation in 96 N. tenuis individuals from 14 locations throughout Japan, based on amplicon sequencing of the 16S ribosomal RNA gene. Nine major bacteria associated with N. tenuis were identified: Rickettsia, two strains of Wolbachia, Spiroplasma, Providencia, Serratia, Pseudochrobactrum, Lactococcus, and Stenotrophomonas. Additionally, a diagnostic PCR analysis for three typical insect reproductive manipulators, Rickettsia, Wolbachia, and Spiroplasma, was performed on a larger sample size (n = 360) of N. tenuis individuals; the most prevalent symbiont was Rickettsia (69.7%), followed by Wolbachia (39.2%) and Spiroplasma (6.1%). Although some symbionts were co-infected, their prevalence did not exhibit any specific tendency, such as a high frequency in specific infection combinations. The infection frequency of Rickettsia was significantly correlated with latitude and temperature, while that of Wolbachia and Spiroplasma was significantly correlated with host plants. The predominance of these bacteria and the absence of obligate symbionts suggested that the N. tenuis microbiome is typical for predatory arthropods rather than sap-feeding insects. Rickettsia and Wolbachia were vertically transmitted rather than horizontally transmitted from the prey. The functional validation of each symbiont would be warranted to develop N. tenuis as a biological control agent.

A comparative approach on the spray and freeze drying of probiotic and Gamma-aminobutyric acid as a single entity: Characterization and evaluation of stability in simulated gastrointestinal conditions

To promote the adequate supplementation of probiotics and bioactives in food formulations, this study aimed to develop synbiotic co-microcapsules containing probiotic strain Lactococcus lactis SKL 13 and bioactive compound, namely γ-Aminobutyric acid (GABA), using spray (at the air inlet and outlet temperatures of 110 ± 2 °C and 50 ± 5 °C, respectively, with a feed flow rate of 2.5 mL min−1) and freeze-drying (at - 40 °C temperature, 1 mbar pressure for 12 h) in a ternary exopolysaccharide (such as maltodextrin, dextran, and inulin in a combination of 8.41, 4.59, and 0.40 g/100 mL, respectively) matrix. The freeze-drying resulted in higher probiotic and GABA encapsulation efficiencies of 95.08 and 90.04%, respectively, than spray drying (probiotics: 93.12% and GABA: 83.46%). The absence of diffraction peaks indicated an amorphous metastable state of microcapsules. Only ∼1% reduction in probiotic count and ∼5% decrease in GABA content were observed in dried capsules after 60 days of storage as well as both the powders showed non-significant (p > 0.05) reduction in probiotic count (2.9 and 1.35 log CFU/mL in intestinal and gastric conditions, respectively) and 80% release of GABA after 240 min of simulated gastrointestinal conditions. The Fourier-transform infrared (FTIR) and scanning electron microscopy (SEM) results further confirmed the proper embedding of probiotics and GABA in the dried microcapsules. The lower moisture content (4.15%), good flowability and higher thermal decomposition peaks at an elevated temperature of 308 °C exhibited by spray-dried powder compared to freeze-dried particles indicated their greater potentiality for high-temperature food applications.

Wild-type Lactococcus lactis producing bacteriocin-like prophage lysins.

Lactococcus is a genus of lactic acid bacteria used in the dairy industry as a starter. Lactococci have been found to produce altogether more than 40 different bacteriocins, ribosomally synthesized antimicrobial proteins. All known Lactococcus spp. bacteriocins belong to classes I and II, which are mainly heat-resistant peptides. No class III bacteriocins, bigger heat-sensitive proteins, including phage tail-like bacteriocins, have been found from the Lactococcus spp. Unlike phage tail-like bacteriocins, prophage lysins have not been regarded as bacteriocins, possibly because phage lysins contribute to autolysis, degrading the host's own cell wall. Wild-type Lactococcus lactis strain LAC460, isolated from spontaneously fermented idli batter, was examined for its antimicrobial activity. We sequenced the genome, searched phage lysins from the culture supernatant, and created knock-out mutants to find out the source of the antimicrobial activity. The strain LAC460 was shown to kill other Lactococcus strains with protease- and heat-sensitive lytic activity. Three phage lysins were identified in the culture supernatant. The genes encoding the three lysins were localized in different prophage regions in the chromosome. By knock-out mutants, two of the lysins, namely LysL and LysP, were demonstrated to be responsible for the antimicrobial activity. The strain LAC460 was found to be resistant to the lytic action of its own culture supernatant, and as a consequence, the phage lysins could behave like bacteriocins targeting and killing other closely related bacteria. Hence, similar to phage tail-like bacteriocins, phage lysin-like bacteriocins could be regarded as a novel type of class III bacteriocins.

Probiotics and human health

Probiotics are living microorganisms which impart beneficial effects to consumers when ingested in a certain quantity. The main probiotic groups include the strains of genera Lactobacillus, Bifidobacterium, Lactococcus and Streptococcus. Probiotics are similar to the naturally found intestinal gut microflora which play an important role in enhancing the human and animal health. Probiotic microorganisms provide various health benefits to human, reduce the blood pressure, decrease the serum cholesterol level, lactose digestion and improve immune response etc. They provide health benefits to the consumers by various mechanisms such as by producing the different postbiotics (bacteriocins, lactic acid), build up the epithelial barrier, fight for binding sites with pathogens on gut wall and thus preventing the infection and enhancing the adhesion to intestinal mucosa. Thus, probiotics having therapeutic values are used for the treatment of various diseases and illness such as allergy, cancer, gastrointestinal disorders, urogenital infections, to reduce obesity, cholesterolaemia and diarrhea. Due to their numerous health benefits, probiotics are gaining more attention among researchers and consumers. More research is needed on probiotics and their action mechanisms which will provide suitable probiotic strain for treatment/prevention of diseases. This review presents the role of probiotic microorganisms in sustaining and improving the health of human being.

Growth differentiation factor 11 delivered by dairy Lactococcus lactis strains modulates inflammation and prevents mucosal damage in a mice model of intestinal mucositis.

Mucositis is an inflammation of the gastrointestinal mucosa that debilitate the quality of life of patients undergoing chemotherapy treatments. In this context, antineoplastic drugs, such as 5-fluorouracil, provokes ulcerations in the intestinal mucosa that lead to the secretion of pro-inflammatory cytokines by activating the NF-κB pathway. Alternative approaches to treat the disease using probiotic strains show promising results, and thereafter, treatments that target the site of inflammation could be further explored. Recently, studies reported that the protein GDF11 has an anti-inflammatory role in several diseases, including in vitro and in vivo results in different experimental models. Hence, this study evaluated the anti-inflammatory effect of GDF11 delivered by Lactococcus lactis strains NCDO2118 and MG1363 in a murine model of intestinal mucositis induced by 5-FU. Our results showed that mice treated with the recombinant lactococci strains presented improved histopathological scores of intestinal damage and a reduction of goblet cell degeneration in the mucosa. It was also observed a significant reduction of neutrophil infiltration in the tissue in comparison to positive control group. Moreover, we observed immunomodulation of inflammatory markers Nfkb1, Nlrp3, Tnf, and upregulation of Il10 in mRNA expression levels in groups treated with recombinant strains that help to partially explain the ameliorative effect in the mucosa. Therefore, the results found in this study suggest that the use of recombinant L. lactis (pExu:gdf11) could offer a potential gene therapy for intestinal mucositis induced by 5-FU.

Metabiotics Signature through Genome Sequencing and In Vitro Inhibitory Assessment of a Novel Lactococcus lactis Strain UTNCys6-1 Isolated from Amazonian Camu-Camu Fruits

Metabiotics are the structural components of probiotic bacteria, functional metabolites, and/or signaling molecules with numerous beneficial properties. A novel Lactococcus lactis strain, UTNCys6-1, was isolated from wild Amazonian camu-camu fruits (Myrciaria dubia), and various functional metabolites with antibacterial capacity were found. The genome size is 2,226,248 base pairs, and it contains 2248 genes, 2191 protein-coding genes (CDSs), 50 tRNAs, 6 rRNAs, 1 16S rRNA, 1 23S rRNA, and 1 tmRNA. The average GC content is 34.88%. In total, 2148 proteins have been mapped to the EggNOG database. The specific annotation consisted of four incomplete prophage regions, one CRISPR-Cas array, six genomic islands (GIs), four insertion sequences (ISs), and four regions of interest (AOI regions) spanning three classes of bacteriocins (enterolysin_A, nisin_Z, and sactipeptides). Based on pangenome analysis, there were 6932 gene clusters, of which 751 (core genes) were commonly observed within the 11 lactococcal strains. Among them, 3883 were sample-specific genes (cloud genes) and 2298 were shell genes, indicating high genetic diversity. A sucrose transporter of the SemiSWEET family (PTS system: phosphoenolpyruvate-dependent transport system) was detected in the genome of UTNCys6-1 but not the other 11 lactococcal strains. In addition, the metabolic profile, antimicrobial susceptibility, and inhibitory activity of both protein-peptide extract (PPE) and exopolysaccharides (EPSs) against several foodborne pathogens were assessed in vitro. Furthermore, UTNCys6-1 was predicted to be a non-human pathogen that was unable to tolerate all tested antibiotics except gentamicin; metabolized several substrates; and lacks virulence factors (VFs), genes related to the production of biogenic amines, and acquired antibiotic resistance genes (ARGs). Overall, this study highlighted the potential of this strain for producing bioactive metabolites (PPE and EPSs) for agri-food and pharmaceutical industry use.

Growth performance of Listeria monocytogenes and background microbiota from mushroom processing environments

Interaction between Listeria monocytogenes and resident background microbiota may occur in food processing environments and may influence the survival of this pathogen in a factory environment. Therefore the aim of this study was to characterize the growth performance of microbiota isolated from the processing environments of frozen sliced mushrooms, and to investigate the competitive performance of L. monocytogenes when co-cultured with accompanying environmental microbiota. Acinetobacter, Enterobacteriaceae, Lactococcus and Pseudomonas were the most prominent background microbiota isolated from the processing environment of frozen sliced mushrooms. All individual microbiota strains were able to grow and form biofilm in filter-sterilized mushroom medium, with the mannitol-consumers Raoultella and Ewingella as top performers, reaching up to 9.6 and 9.8 log CFU/mL after 48 h incubation at room temperature. When L. monocytogenes mushroom isolates were co-cultured with the microbiota strains, L. monocytogenes counts ranged from 7.6 to 8.9 log CFU/mL after 24 h of incubation, while counts of the microbiota strains ranged from 5.5 to 9.0 log CFU/mL. Prolonged incubation up to 48 h resulted in further increase of L. monocytogenes counts when co-cultured with non-acidifying species Pseudomonas and Acinetobacter reaching 9.1 to 9.2 log CFU/mL, while a decrease of L. monocytogenes counts reaching 5.8 to 7.7 log CFU/mL was observed in co-culture with Enterobacteriaceae and acidifying Lactococcus representatives. In addition, L. monocytogenes grew also in spent mushroom media of the microbiota strains, except in acidified spent media of Lactococcus strains. These results highlight the competitive ability of L. monocytogenes during co-incubation with microbiota in fresh and in spent mushroom medium, indicative of its invasion and persistence capacity in food processing factory environments.

Screening of lactic acid bacteria strains isolated from Iranian traditional dairy products for GABA production and optimization by response surface methodology

A total of 50 lactic acid bacteria (LAB) isolates from Iranian traditional dairy products (Motal and Lighvan cheeses, and artisanal yogurt) were screened for gamma-aminobutyric acid (GABA) production. Firstly, a rapid colorimetric test was performed to evaluate the glutamate decarboxylase (GAD) activity among the LAB isolates examined. Thin layer chromatography (TLC) was then performed on selected strains to identify isolates with high/moderate GABA producing capacity, and a GABase micro-titer plate assay was employed to quantify GABA. Finally, two Lactococcus (Lac.) lactis strains were selected for GABA production optimization via Response Surface Methodology (RSM) following Central Composite Design (CCD). Forty-one out of the 50 isolates showed GAD activity according to the colorimetric assay. Eight isolates displayed strong GAD activity, while nine showed no activity; low to moderate GAD activity was scored for all other isolates. GABA production was confirmed by TLC in all isolates with high GAD activity and in four selected among isoaltes with moderate activity. Among the Lactococcus strains tested, Lac. lactis 311 and Lac. lactis 491 were the strongest GABA producers with amounts of 3.3 and 1.26 mM, respectively. These two strains were subjected to GABA production optimization applying RSM and CCD on three key variables: Monosodium glutamate concentration (MSG) (between 25 and 150 mM), incubation temperature (between 25 and 37 °C), and pH (between 4.0 and 5.0). Optimal conditions for GABA production by Lac. lactis 311 and Lac. lactis 491 of temperature, pH and MSG concentration were, respectively, 35.4 and 30 °C, pH 4.5 and 4.6, and MSG concentration of 89 and 147.4 mM, respectively. Under the above conditions, the amount of GABA produced by Lac. lactis 311 and Lac. lactis 491 was 0.395 and 0.179 mg/mL, respectively. These strains and the optimal culture conditions determined in this study could be used for the biotechnological production of GABA or applied in food fermentations for the development of naturally GABA-enriched foods.

GROWTH STIMULATION OF Lactococcus lactis sps BY XYLOOLIGOSACCHARIDES PRODUCED USING SINGLE FERMENTATIVE STEP FROM LIGNOCELLULOSIC WASTE BY Massilia Timonae B2YR

As a source of xylan, lignocellulose waste such as wheat bran and aquatic weed Pistia stratiotes were employed, together with potassium nitrate as a nitrogen source, which were optimized using the Box- Behnken design to increase the production of xylooligosaccharides. The yield of xylooligosaccharides increased to 2.05- fold in the optimized medium than non-optimized medium. The produced xylooligosaccharides showed a peak at 890-900 cm-1 confirming the presence of β-glycosidic linkages when detected by Fourier Transform Infra-Red (FT IR). The prebiotic effect of produced xylooligosaccharides was studied on two strains named Lactococcus lactis Cl MN515342 and Lactococcus lactis Cp MN515343 isolated from curd samples. The growth of these Lactococcus strains was found to be stimulated. A log Colony Forming Unit/ml value of 7.813 and 8.146 for Cp and Cl was observed in a medium incorporated with XOS. The Short-Chain Fatty Acid (SCFA) production by Lactococcus lactis in a medium incorporated with xylooligosaccharides was detected by GCMS. The use of lignocellulosic waste for the production of xylooligosaccharides reduces the production cost. The xylooligosaccharides produced by Massilia timonae has shown promising result in stimulating the growth of Lactococcus lactis. Thus, XOS can be incorporated in curd or Yoghurt along with the Lactococcus lactis and used as synbiotic food.