Cell Growth Density and Nisin A Activity of the Indigenous Lactococcus lactis subsp. cremoris M78 Costarter Depend Strongly on Inoculation Levels of a Commercial Streptococcus thermophilus Starter in Milk: Practical Aspects for Traditional Greek Cheese Processors

2020 ◽  
Vol 83 (3) ◽  
pp. 542-551
Author(s):  
JOHN SAMELIS ◽  
ATHANASIA KAKOURI
Keyword(s):  
Lactococcus Lactis ◽  
Streptococcus Thermophilus ◽  
Raw Milk ◽  
Starter Cultures ◽  
Competitive Growth ◽  
Population Densities ◽  
Relative Population ◽  
Lactococcus Lactis Subsp ◽  
Pure Cultures

ABSTRACT Mixed thermophilic and mesophilic commercial starter cultures (CSCs), particularly those including Streptococcus thermophilus as a primary milk acidifier, have been found to reduce growth and counteract in situ nisin A (NisA+) antilisterial effects by the novel, indigenous Lactococcus lactis subsp. cremoris M78 costarter in traditional Graviera thermized milk cheese curds. Therefore, this model challenge study evaluated growth and in situ NisA+ activity of strain M78 in coculture with S. thermophilus ST1 singly in sterilized raw milk (SRM). Strain ST1, derived from a CSC for cheese, was challenged at two inoculation levels (5 and 7 log CFU/mL) in SRM against 6 and 3 log CFU/mL of strain M78 and Listeria monocytogenes, respectively. Pure cultures of each strain and cocultures of strain ST1 with the CSC L. lactis LL2, in replacement of strain M78, served as controls. At the high (7-log) inoculation level, the rapid, competitive growth (>9.3 log CFU/mL) of S. thermophilus ST1 reduced growth of both L. lactis by at least 10-fold; the industrial strain LL2 retained slightly higher relative population densities (7.4 to 9.1%) than the wild NisA+ strain M78 (3.8 to 5.6%) after 6 h at 37°C, followed by an additional 66 h of incubation at 22°C. In full contrast, at the low (5-log) inoculation level, S. thermophilus ST1 failed to predominate in SRM at 6 h; thus, the starter lactic acid bacteria populations were reversed in favor of L. lactis. Notably, strain M78 retained higher relative population densities (83.0 to 90.1%) than the CSC strain LL2 (80.3 to 85.2%) at 22°C. Moreover, at the 5-log ST1 level, the direct and deferred in situ NisA+ activities of strain M78 were at similar levels with its pure culture with L. monocytogenes in SRM, whereas at the 7-log ST1 level, the respective NisA+ effects were counteracted. Hence, 10- to 100-fold lowered inoculation levels of CSC S. thermophilus are required to enhance the performance of the M78 costarter in traditional Greek cheese technologies. HIGHLIGHTS

Growth, nisA Gene Expression, and In Situ Activity of Novel Lactococcus lactis subsp. cremoris Costarter Culture in Commercial Hard Cheese Production

2017 ◽  
Vol 80 (12) ◽  
pp. 2137-2146 ◽  
Author(s):  
Dimitrios Noutsopoulos ◽  
Athanasia Kakouri ◽  
Eleftheria Kartezini ◽  
Dimitrios Pappas ◽  
Efstathios Hatziloukas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lactococcus Lactis ◽  
Starter Culture ◽  
Fold Increase ◽  
Raw Milk ◽  
Good Growth ◽  
Lactococcus Lactis Subsp ◽  
Quantitative Expression ◽  
Hard Cheese

ABSTRACT This study evaluated in situ expression of the nisA gene by an indigenous, nisin A–producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (>8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A–mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

scholarly journals Importância das Bactérias Ácido Láticas e não Starter (NSLAB) na Tecnologia de Produção dos Derivados Lácteos

2020 ◽  
Vol 24 (4) ◽  
pp. 348-352
Author(s):  
Maria Tereza Pereira ◽  
Elsa Helena Walter de Santana ◽  
Joice Sifuentes dos Santos
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactococcus Lactis ◽  
Lactobacillus Casei ◽  
Streptococcus Thermophilus ◽  
Raw Milk ◽  
Fermented Milk ◽  
Starter Cultures ◽  
Bacillus Spp ◽  
Lactobacillus Spp

Produtos lácteos fermentados contêm bactérias ácido lácticas (BAL), naturalmente presentes ou adicionadas na matriz láctea como culturas iniciadoras (starters), contribuindo com aroma, textura, valor nutricional e segurança microbiológica. Lactobacillus spp., Streptococcus spp., Lactococcus spp. e Leuconostoc spp. são utilizados como culturas starters em laticínios. As BAL podem ser classificadas em mesofílicas (ex Lactococcus lactis) e termofílicas (ex Streptococcus thermophilus), e de acordo com seus metabólitos de fermentação em homofermentativas (ácido lático) e heterofermentativas (ácido lático, dióxido de carbono, diacetil e outros compostos flavorizantes). Entre as BAL há um grupo de bactérias lácticas que não fazem parte da cultura láctica (non starter lactic acid bacteria - NSLAB), que são oriundas do leite cru, do ambiente de ordenha ou da indústria formando biofilmes. As NSLAB são representadas por espécies heterofermentativas de lactobacilos mesofílicos como Lactobacillus casei spp., L. paracasei spp., L. rhamnosus spp. e L. plantarum spp., e ainda por Pediococcus spp., Leuconostoc spp. e Micrococcus spp. NSLAB termoduricas como Bacillus spp. também são relatadas. As NSLAB em queijos podem ajudar a desenvolver sabor e aroma, porém também são associadas aos defeitos em queijos e leites fermentados. Problemas como odores estranhos, sabor amargo ou muito ácido, perda de viscosidade, perda de coloração, estufamento e formação de gás são associados com a presença e contaminação por NSLAB. Assim, as BAL são importantes micro-organismos na indústria láctea, garantindo sabores e aromas aos derivados. Já a presença de NSLAB podem ser associados com defeitos em queijos e leites fermentados, sendo um problema na indústria beneficiadora.   Palavras-chave: Característica Sensorial. Leites Fermentados. Queijo. Textura.                       Abstract Fermented dairy products contain acid bacteria (BAL) naturally present or added to the dairy matrix as starter cultures (starters), contributing to aroma, texture, nutritional value and microbiological safety. Lactobacillus spp., Streptococcus spp., Lactococcus spp. and Leuconostoc spp. are used as starter dairy crops. As BAL it can be classified as mesophilic (ex: Lactococcus lactis) and thermophilic (ex: Streptococcus thermophilus), and agree with its fermentation metabolites in homofermentative (lactic acid) and heterofermentative (lactic acid, carbon dioxide, diacetyl and other flavorings). Among the BAL, there is a group of lactic bacteria that are not part of the dairy culture (non-initiating lactic acid bacteria - NSLAB) that originate from raw milk, the milking environment or the biofilm-forming industry. NSLAB is represented by heterofermentative species of mesophilic lactobacilli such as Lactobacillus casei spp., L. paracasei spp., L. rhamnosus spp. and L. plantarum spp., and also by Pediococcus spp., Leuconostoc spp. and Micrococcus spp. Termoduric NSLAB such as Bacillus spp. are also related. NSLAB in cheeses may help develop flavor and aroma, and they are also associated with defects in fermented cheeses and milks. Problems such as strange odors, bitter or very acidic taste, loss of viscosity, loss of color, establishment and gas training are associated with the presence and contamination by NSLAB. Thus,  BALs are important microorganisms in the dairy industry, contributing to the dairy flavors and aromas. The presence of NSLAB, on the other hand, can be associated with defects in fermented milk and cheese, being a problem in the processing industry.   Keywords: Cheese. Fermented Milk. Sensory Characteristic. Texture.

scholarly journals Effect of pasteurization and starter cultures on physicochemical and microbiological properties of costeño cheese

2016 ◽  
Vol 69 (2) ◽  
pp. 8007-8014 ◽  
Author(s):  
José G. Serpa ◽  
Tulia I. Pérez ◽  
Elvis J. Hernández
Keyword(s):  
Lactococcus Lactis ◽  
Control Sample ◽  
Raw Milk ◽  
Starter Cultures ◽  
Sensorial Analysis ◽  
Pasteurized Milk ◽  
Randomized Design ◽  
Completely Randomized Design ◽  
And Control ◽  
Microbiological Properties

The effect of pasteurization and starter cultures on physicochemical, microbiological and sensorial characteristics of costeño cheese was determined. A completely randomized design was conducted, three treatments (T) and three replicates: Treatment 1 (T1): cheese manufactured with pasteurized milk without starter cultures, Treatment 2 (T2): cheese manufactured with pasteurized milk with Lactococcus lactis and Lactococcus cremoris (1:1) and Treatment 3 (T3): cheese manufactured with pasteurized milk with Lactococcus lactis, Lactococcus cremoris and Streptococcus thermophillus(0.5:0.5:1). Treatments were compared to a control sample that was prepared with raw milk without starter cultures. Concentration of 1.5% (v/v) of culture was used in relation to the amount of used milk in each treatment. Moisture content was higher in all treatments compared to the control and protein and fat content were significantly lower. Acidity was significantly higher in samples from T2 y T3 compared to T1 and control, due to the metabolism of starter cultures. Total coliforms, yeast and mold counts showed a significant reduction due to pasteurization process in all treatments. Regarding sensorial analysis, hedonic test showed a greater preference in cheese manufactured with T2 (P<0.05). There were no significant preferences between T1, T3 and control. Additionally, yield was significantly higher with T1 (22%) and T3 (23%) compared to control.

scholarly journals Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Content Type ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits

ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

Persistence of Escherichia coli O157:H7 in Dairy Fermentation Systems

1998 ◽  
Vol 61 (12) ◽  
pp. 1602-1608 ◽  
Author(s):  
SEAN S. DINEEN ◽  
KAZUE TAKEUCHI ◽  
JANE E. SOUDAH ◽  
KATHRYN J. BOOR
Keyword(s):  
Escherichia Coli ◽  
Lactococcus Lactis ◽  
Dairy Products ◽  
Starter Cultures ◽  
Escherichia Coli O157 ◽  
Potential Health ◽  
E Coli ◽  
Lactococcus Lactis Subsp ◽  
Fermented Dairy Products ◽  
Fermented Dairy

We examined (i) the persistence of Escherichia coli O157:H7 as a postpasteurization contaminant in fermented dairy products; (ii) the ability of E. coli O157:H7 strains with and without the general stress regulatory protein, RpoS, to compete with commercial starter cultures in fermentation systems; and (iii) the survival of E. coli O157:H7 in the yogurt production process. In commercial products inoculated with 103 CFU/ml, E. coli O157:H7 was recovered for up to 12 days in yogurt (pH 4.0), 28 days in sour cream (pH 4.3), and at levels &gt;102 CFU/ml at 35 days in buttermilk (pH 4.1). For the starter culture competition trials, the relative inhibition of E. coli O157:H7 in the experimental fermentation systems was, in decreasing order, thermophilic culture mixture, Lactobacillus delbrueckii subsp. bulgaricus R110 alone, Lactococcus lactis subsp. lactis D280 alone, Lactococcus lactis subsp. cremoris D62 alone, and Streptococcus thermophilus C90 alone showing the least inhibition. Recovery of the rpoS mutant was lower than recovery of its wild-type parent by 72 h or earlier in the presence of individual starter cultures. No E. coli O157:H7 were recovered after the curd formation step in yogurt manufactured with milk inoculated with 105 CFU/ml. Our results show that (i) postprocessing entry of E. coli O157:H7 into fermented dairy products represents a potential health hazard; (ii) commercial starter cultures differ in their ability to reduce E. coli O157:H7 CFU numbers in fermentation systems; and (iii) the RpoS protein appears to most effectively contribute to bacterial survival in the presence of conditions that are moderately lethal to the cell.

scholarly journals Autochthonous Starter Effect on the Microbiological, Physicochemical and Sensorial Characteristics of Moroccan Goat’s Milk Cheeses

2014 ◽  
Vol 4 (1) ◽  
pp. 343-351
Author(s):  
Ouiam El Galiou ◽  
Juan Antonio Centeno ◽  
Said Zantar ◽  
Mohammed Bakkali ◽  
Javier Carballo ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Starter Cultures ◽  
Lactobacillus Paracasei ◽  
Sensory Characteristics ◽  
Total Solids ◽  
Lactococcus Lactis Subsp ◽  
Ph Values

The effect of adding four Lactococcus lactis subsp. Lactis, Lactococcus lactis subsp. lactis Bv. diacetylactis and Lactobacillus paracasei autochthonous strains as single starters (A and B) on the microbiological, physicochemical and sensory characteristics of Moroccan goat’s milk cheese was investigated. Batches with a commercial starter (C) and with raw goat’s milk without starter (D) were also made as controls. The results indicated that the different starter cultures used significantly affected the compositional characteristics of Moroccan goat’s cheese. At the first day of ripening, the pH values of cheeses manufactured with any type of starter were significantly higher than those made without starter. No significant differences (P >0.05) in values of total solids (T.S.), proteins, ash, FAT and lactose content were found between any of the batches after 7 days of ripening. The autochthonous starters in Moroccan cheesemaking were successful at reducing the levels of coliforms earlier in ripening than in cheeses made without starter and even in those made with the commercial starter. These results suggest that the combined activity of L. Lactis subsp. lactis, L. Lactis. subsp. Lactis bv. diacetylactis and L. paracasei, produces cheese with good sensory characteristics.

scholarly journals Unleashing natural competence inLactococcus lactisby induction of the competence regulator ComX

2017 ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits ◽  
Derivatives Of

AbstractIn biotechnological work horses likeStreptococcus thermophilusandBacillus subtilisnatural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starterLactococcus lactisnatural competence has not been established to date. However,in silicoanalysis of complete genome sequences of 43L. lactisstrains revealed complete late-competence gene-sets in 2L. lactissubspeciescremorisstrains (KW2 and KW10) and 8L. lactissubspecieslactisstrains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulatorcomXinL. lactissubsp.lactisKF147 resulted in the induction of expression of the canonical competence regulon, and elicited a state of natural competence in this strain. By contrast,comXexpression inL. lactisNZ9000, predicted to encode an incomplete competence gene-set, failed to induce natural competence. Moreover, mutagenesis of thecomEA-ECoperon in strain KF147, abolished thecomXdriven natural competence, underpinning the involvement of the competence machinery. Finally, introduction of nisin-induciblecomXexpression intonisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence also in these strains, expanding this phenotype to otherL. lactisstrains of both subspecies.Significance statementSpecific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the speciesLactococcus lactisare very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end-product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria, and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

Monitoring growth compatibility and bacteriocin gene transcription of adjunct with starter lactic acid bacteria strains in milk

2020 ◽  
Author(s):  
Stamatia Asimakoula ◽  
Katerina Giaka ◽  
Christos Fanitsios ◽  
Athanasia Kakouri ◽  
Elpiniki Vandera ◽  
...  
Keyword(s):  
Real Time ◽  
Streptococcus Thermophilus ◽  
Antagonistic Activity ◽  
Starter Cultures ◽  
Real Time Quantitative Pcr ◽  
Reverse Transcription Pcr ◽  
Combination Treatments ◽  
Gene Transcripts ◽  
Bacteriocin Gene

When developing protective starter cultures for application in cheese technologies, monitoring growth interactions between starter and adjunct LAB species and in situ expression of bacteriocin genes in the mixtures, are crucial. This study firstly aimed to monitor growth of mixed LAB strain populations during milk model fermentations by microbial counts and real-time quantitative PCR (qPCR). The primary starter Streptococcus thermophilus ST1 and costarter Lactococcus lactis subsp. cremoris M78 strains served as the basic starter composite co-inoculated in all milk treatments. Adjunct bacteriocinogenic Enterococcus faecium strains KE82 and GL31 and a ripening Lactiplantibacillus plantarum H25 strain were added separately to the starter composite resulting in four LAB combination treatments. The second aim was to quantify gene transcripts of nisin and enterocins B and A synthesized by M78, KE82 and GL31, respectively, by real-time reverse transcription PCR (RT-qPCR) and to detect the in situ antilisterial effects of the cocultures. Adjunct LAB strains showed growth compatibility with the starter, since all of them exhibited a 2 to 3 log units increase in their population levels, compared to their initial inoculation levels, with ST1 prevailing in all treatments. KE82 grew more competitively than GL31 whereas cocultures with KE82 displayed the strongest in situ antilisterial activity. Nisin gene expression levels were higher at the exponential phase of microbial growth in all treatments. Finally, expression of nisin and enterocins A and B genes was interrelated indicating an antagonistic activity.

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