Adaptive laboratory evolution as a means to generate Lactococcus lactis strains with industrially relevant traits – Characterization of isolates with improved thermotolerance and ability to autolyze

2021 ◽  
Author(s):  
Robin Dorau ◽  
Jun Chen ◽  
Jianming Liu ◽  
Peter Ruhdal Jensen ◽  
Christian Solem
Keyword(s):  
Lactococcus Lactis ◽  
Elevated Temperatures ◽  
Starter Culture ◽  
High Growth ◽  
Starter Cultures ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Cheese Ripening ◽  
Underlying Causes

Lactococcus lactis subsp. lactis ( L. lactis ) is a model lactic acid bacterium and one of the main constituents of the mesophilic cheese starter used for producing soft or semi-hard cheeses. Most dairy L. lactis strains grow optimally at around 30°C and are not particularly well-adapted to the elevated temperatures (37-39°C), which they are often exposed to during cheese production. To overcome this challenge, we used Adaptive Laboratory Evolution (ALE) in milk, using a setup where the temperature was gradually increased over time, and isolated two evolved strains (RD01 and RD07) better able to tolerate high growth temperatures. One of these, strain RD07, was isolated after one and a half years of evolution (400 generations) and efficiently acidified milk at 41°C, which has not been reported for industrial L. lactis strains until now. Moreover, RD07 appeared to autolyze 2-3 times faster than its parent strain, which is another highly desired property of dairy lactococci and rarely observed in the lactis subspecies used in this study. Model cheese trials indicated that RD07 could potentially accelerate cheese ripening. Transcriptomics analysis revealed the potential underlying causes responsible for the enhanced growth at high temperatures for the mutants. These included downregulation of the pleiotropic transcription factor CodY and overexpression of genes, which most likely lowered the guanidine nucleotide pool. Cheese trials at ARLA Foods using RD01 blended with the commercial Flora Danica starter culture, including a 39.5°C cooking step, revealed better acidification and flavor formation than the pure starter culture. Importance In commercial mesophilic starter cultures, L. lactis is generally more thermotolerant than L. cremoris , whereas L. cremoris is more prone to autolysis, which is the key to flavor and aroma formation. In this study, we find that adaptation to higher thermotolerance can improve autolysis. Using whole-genome sequencing and RNA-sequencing, we attempt to determine the underlying reason for the observed behavior. In terms of dairy applications, there are obvious advantages associated with using L. lactis strains with high thermotolerance, as these are less affected by curd cooking, which generally hampers the performance of the mesophilic starter. Cheese ripening, the most costly part of cheese manufacturing, can be reduced using autolytic strains. Thus, the solution presented here could simplify starter cultures, make the cheese manufacturing process more efficient, and enable novel types of harder cheese variants.

A Food-Grade System for Production of Pediocin PA-1 in Nisin-Producing and Non–Nisin-Producing Lactococcus lactis Strains: Application To Inhibit Listeria Growth in a Cheese Model System

2007 ◽  
Vol 70 (11) ◽  
pp. 2512-2517 ◽  
Author(s):  
C. REVIRIEGO ◽  
L. FERNÁNDEZ ◽  
J. M. RODRÍGUEZ
Keyword(s):  
Antimicrobial Activity ◽  
Lactococcus Lactis ◽  
Model System ◽  
Starter Cultures ◽  
Listeria Innocua ◽  
Heterologous Production ◽  
Technological Properties ◽  
Food Grade ◽  
Cheese Ripening ◽  
Cheese Model

Food-grade heterologous production of pediocin PA-1 in nisin-producing and non–nisin-producing Lactococcus lactis strains, previously selected because of their technological properties for cheese making, was achieved. Plasmid pGA1, which contains the complete pediocin operon under the control of the strong P32 promoter and is devoid of any antibiotic marker, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis+). Transformation of L. lactis ESI 515 with pGA1 did not affect its ability to produce nisin. The antimicrobial activity of the pediocin-producing transformants on the survival of Listeria innocua SA1 during cheese ripening was also investigated. Cheeses were manufactured from milk inoculated with 1% of the lactic culture and with or without approximately 4 log CFU/ml of the Listeria strain. L. lactis ESI 153, L. lactis ESI 515, and their transformants (L. lactis GA1 and GA2, respectively) were used as starter cultures. At the end of the ripening period, counts of L. innocua in cheeses made with the bacteriocin-producing lactococcal strains were below 50 CFU/g in the L. lactis GA1 cheeses and below 25 CFU/g in the L. lactis GA2 ones, compared with 3.7 million CFU/g for the controls without nisin or pediocin production.

Changes in Galactose and Lactic Acid Content of Sweet Whey during Storage

2004 ◽  
Vol 67 (2) ◽  
pp. 403-406 ◽  
Author(s):  
R. D. RAO ◽  
W. L. WENDORFF ◽  
K. SMITH
Keyword(s):  
Lactic Acid ◽  
Lactococcus Lactis ◽  
Lactic Acid Production ◽  
Starter Culture ◽  
Storage Conditions ◽  
Starter Cultures ◽  
Lactobacillus Helveticus ◽  
Lactobacillus Delbrueckii ◽  
The Other ◽  
Time Period

Whey is often stored or transported for a period of time prior to processing. During this time period, galactose and lactic acid concentrations may accumulate, reducing the quality of spray-dried whey powders in regard to stickiness and agglomeration. This study surveyed industry samples of Cheddar and mozzarella cheese whey streams to determine how galactose and lactic acid concentrations changed with storage at appropriate (4°C) and abuse (37.8°C) temperatures. Samples stored at 4°C did not exhibit significant increases in levels of lactic acid or galactose. Mozzarella whey accumulated the greatest amount of galactose and lactic acid with storage at 37.8°C. Whey samples derived from cheese made from single strains of starter culture were also evaluated to determine each culture's contribution to galactose and lactic acid production. Starter cultures evaluated included Streptococcus salivarius ssp. thermophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, Lactococcus lactis ssp. cremoris, and Lactococcus lactis ssp. lactis. Whey derived from L. helveticus accumulated a significantly greater amount of lactic acid upon storage at 37.8°C as compared with the other cultures. Galactose accumulation was significantly decreased in whey from L. lactis ssp. lactis stored at 37.8°C in comparison with the other cultures. Results from this study indicate that proper storage conditions (4°C) for whey prevent accumulation of galactose and lactic acid while the extent of accumulation during storage at 37.8°C varies depending on the culture(s) used in cheese production.

Genetic characterization of partial lactose-fermenting revertants from lactose-negative mutants of lactococci

1991 ◽  
Vol 37 (4) ◽  
pp. 281-286 ◽  
Author(s):  
R. P. Sinha
Keyword(s):  
Key Words ◽  
Plasmid Dna ◽  
Genetic Information ◽  
Genetic Characterization ◽  
Skim Milk ◽  
Starter Cultures ◽  
Chromosomal Gene ◽  
Cheese Ripening ◽  
Lactose Fermentation

Partial lactose-positive (Lac+) revertants, designated RS0121 and RS033, were isolated from lactose-negative (Lac−) mutants of Lactococcus lactis subsp. lactis C2 and ML3, respectively. These revertants were found to coagulate 10% reconstituted skim milk, but the rate of lactose fermentation was much slower than that of their parental Lac+ strains. Such partial Lac+ revertants were also isolated from Lac− mutants of L. lactis subsp. Lactis SL712, SH4109, and SL763 and of L. lactis subsp. cremoris ML1 and SC607, showing a generalized phenomenon. The Lac+ characteristic of the revertant strains was conjugally transferred to Lac− variants. When the plasmid profiles of the Lac− variants and their partial Lac+ revertants and transconjugants were analyzed, there were no detectable differences in the plasmid bands. A strain with no plasmids, MG1363, also yielded partial Lac+ revertants, which were devoid of all plasmids. In addition, the Lac+ phenotype of one such revertant, designated RS101, was transferable to other Lac− strains by conjugation. The results indicate that the genetic information for slow lactose metabolism in such partial Lac+ revertants is mediated through chromosomal gene(s) and is under the influence of some suppressor. These observations also suggest the possibility of utilizing this method for isolating slow acid producing variants, which could be used as adjuncts with regular cheese starter cultures for accelerated cheese ripening. Key words: lactococci, lactose-negative mutants, plasmid DNA, lactose-fermenting revertants.

scholarly journals Detection and Characterization of Streptococcus thermophilus Bacteriophages by Use of the Antireceptor Gene Sequence

2005 ◽  
Vol 71 (10) ◽  
pp. 6096-6103 ◽  
Author(s):  
Ana G. Binetti ◽  
Beatriz Del Río ◽  
M. Cruz Martín ◽  
Miguel A. Álvarez
Keyword(s):  
Starter Culture ◽  
Dairy Industry ◽  
Pcr Amplification ◽  
Variable Region ◽  
Streptococcus Thermophilus ◽  
Resistance Mechanisms ◽  
Starter Cultures ◽  
Phage Types ◽  
Specific Pcr

ABSTRACT In the dairy industry, the characterization of Streptococcus thermophilus phage types is very important for the selection and use of efficient starter cultures. The aim of this study was to develop a characterization system useful in phage control programs in dairy plants. A comparative study of phages of different origins was initially performed based on their morphology, DNA restriction profiles, DNA homology, structural proteins, packaging mechanisms, and lifestyles and on the presence of a highly conserved DNA fragment of the replication module. However, these traditional criteria were of limited industrial value, mainly because there appeared to be no correlation between these variables and host ranges. We therefore developed a PCR method to amplify VR2, a variable region of the antireceptor gene, which allowed rapid detection of S. thermophilus phages and classification of these phages. This method has a significant advantage over other grouping criteria since our results suggest that there is a correlation between typing profiles and host ranges. This association could be valuable for the dairy industry by allowing a rational starter rotation system to be established and by helping in the selection of more suitable starter culture resistance mechanisms. The method described here is also a useful tool for phage detection, since specific PCR amplification was possible when phage-contaminated milk was used as a template (detection limit, 105 PFU ml−1).

scholarly journals Use of a Genetically Enhanced, Pediocin-Producing Starter Culture, Lactococcus lactis subsp. lactis MM217, To Control Listeria monocytogenes in Cheddar Cheese

1998 ◽  
Vol 64 (12) ◽  
pp. 4842-4845 ◽  
Author(s):  
Nurliza Buyong ◽  
Jan Kok ◽  
John B. Luchansky
Keyword(s):  
Listeria Monocytogenes ◽  
Lactococcus Lactis ◽  
Manufacturing Process ◽  
Acid Production ◽  
Starter Culture ◽  
Cheddar Cheese ◽  
Starter Cultures ◽  
Significant Potential ◽  
Ph Values

ABSTRACT Cheddar cheese was prepared with Lactococcus lactissubsp. lactis MM217, a starter culture which contains pMC117 coding for pediocin PA-1. About 75 liters of pasteurized milk (containing ca. 3.6% fat) was inoculated with strain MM217 (ca. 106 CFU per ml) and a mixture of three Listeria monocytogenes strains (ca. 103 CFU per ml). The viability of the pathogen and the activity of pediocin in the cheese were monitored at appropriate intervals throughout the manufacturing process and during ripening at 8°C for 6 months. In control cheese made with the isogenic, non-pediocin-producing starter culture L. lactis subsp. lactis MM210, the counts of the pathogen increased to about 107 CFU per g after 2 weeks of ripening and then gradually decreased to about 103 CFU per g after 6 months. In the experimental cheese made with strain MM217, the counts of L. monocytogenes decreased to 102 CFU per g within 1 week of ripening and then decreased to about 10 CFU per g within 3 months. The average titer of pediocin in the experimental cheese decreased from approximately 64,000 arbitrary units (AU) per g after 1 day to 2,000 AU per g after 6 months. No pediocin activity (<200 AU per g) was detected in the control cheese. Also, the presence of pMC117 in strain MM217 did not alter the cheese-making quality of the starter culture, as the rates of acid production, the pH values, and the levels of moisture, NaCl, and fat of the control cheese and the experimental cheese were similar. Our data revealed that pediocin-producing starter cultures have significant potential for protecting natural cheese against L. monocytogenes.

scholarly journals Lactococcus lactisDiversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing ofepsD

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Cyril A. Frantzen ◽  
Hans Petter Kleppen ◽  
Helge Holo
Keyword(s):  
Lactococcus Lactis ◽  
Starter Culture ◽  
Amplicon Sequencing ◽  
Core Gene ◽  
Starter Cultures ◽  
Content Type ◽  
Genome Analyses ◽  
Dairy Starter Cultures ◽  
Rotation Strategy ◽  
Cheese Production

ABSTRACTUndefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures ofLactococcus lactisand leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity ofLactococcus lactisstrains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21L. lactissubsp. lactisand 28L. lactissubsp.cremorislineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing usingpurR, a core gene, andepsD, present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter cultureLactococcus lactisand revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk.IMPORTANCEIn contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of more robust starter cultures and assist in maintaining the efficiency and stability of the production process by ensuring the presence of key bacteria that are important to the characteristics of the product.

scholarly journals Characterization of a Leuconostoc Bacteriophage Infecting Flavor Producers of Cheese Starter Cultures

2012 ◽  
Vol 78 (18) ◽  
pp. 6769-6772 ◽  
Author(s):  
Hans Petter Kleppen ◽  
Ingolf F. Nes ◽  
Helge Holo
Keyword(s):  
Leuconostoc Mesenteroides ◽  
Starter Culture ◽  
Starter Cultures ◽  
Content Type ◽  
Culture Isolate

ABSTRACTDairy siphovirus φLmd1, which infects starter culture isolateLeuconostoc mesenteroidessubsp.dextranicumA1, showed resistance to pasteurization and was able to grow on 3 of the 4 commercial starter cultures tested. Its 26,201-bp genome was similar to that ofLeuconostocphage of vegetable origin but not to those of dairy phages infectingLactococcus.

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