Mesophilic Starter Research Articles

Continuous mixed strain mesophilic lactic starter production in supplemented whey permeate medium using immobilized cell technology

Continuous mixed strain mesophilic lactic starter production in supplemented whey permeate medium using immobilized cell technology

Purification and Characterization of Cystathionine (beta)-Lyase from Lactococcus lactis subsp. cremoris B78 and Its Possible Role in Flavor Development in Cheese

An enzyme that degrades sulfur-containing amino acids was purified from Lactococcus lactis subsp. cremoris B78; this strain was isolated from a mixed-strain, mesophilic starter culture used for the production of Gouda cheese. The enzyme has features of a cystathionine (beta)-lyase (EC 4.4.1.8), a pyridoxal-5(prm1)-phosphate-dependent enzyme involved in the biosynthesis of methionine and catalyzing an (alpha),(beta)-elimination reaction. It is able to catalyze an (alpha),(gamma)-elimination reaction as well, which in the case of methionine, results in the production of methanethiol, a putative precursor of important flavor compounds in cheese. The native enzyme has a molecular mass of approximately 130 to 165 kDa and consists of four identical subunits of 35 to 40 kDa. The enzyme is relatively thermostable and has a pH optimum for activity around 8.0; it is still active under cheese-ripening conditions, viz., pH 5.2 to 5.4 and 4% (wt/vol) NaCl. A possible essential role of the enzyme in flavor development in cheese is suggested.

Detection of dairy Leuconostoc strains using the polymerase chain reaction

This paper reports the design of a Leuconostoc-specific oligonucleotide based on 16S rRNA sequence data. When this oligonucleotide was used in a polymerase chain reaction (PCR) in conjunction with an oligonucleotide to a conserved region of the 16S rRNA sequence, a Leuconostoc-specific PCR product of approximately 470 bp was produced. The use of a second oligonucleotide to a conserved region allowed the production of an approximately 350 bp product in all PCRs, acting as a positive control. The PCR procedure described was particularly useful for detecting the presence of Leuconostoc in mixed mesophilic starter cultures. The Leuconostoc-specific oligonucleotide was used also as a specific hybridization probe.

The quality of white-brined cheese from goat's milk made with different starters

Microbiological assessment, biochemical studies and organoleptical evaluation of cheese made from pasteurized goat's milk with four different starter cultures were compared. Cheese made with starter B ( Lactococcus lactis subsp. lactis + Lactobacillus casei subsp. casei + Leuconostoc mesenteroides subsp. cremoris 6:2:2), C ( Lact. lactis subsp. lactis + Lb. casei subsp. casei + Enterococcus durans 6:2:2) or D ( Lact. lactis subsp. lactis + Lb. casei subsp. casei + E. durans + Leuc. mesenteroides subsp. cremoris 6:2:1:1) had lower ( P Streptococcus thermophilus + Lactobacillus delbrueckii subsp. bulgaricus 1:1) and this coincided with higher ( P P P E. durans in the fresh cheese. Cheeses made with starter C or D were graded higher than cheese made with starter A or B. These results suggest that mesophilic starter cultures concurrently used with E. durans might be considered appropriate for the manufacture of white-brined cheese from pasteurized goat's milk.

Impedance measurement of growth of lactic acid bacteria in dairy cultures with honey addition

Abstract The effect of honey addition (0, 1, 3, 5 or 10%, wt/vol) to skim milk on the growth of Lactobacillus acidophilus and a mesophilic starter culture (consisting of Lactococcus lactis , subsp. lactis, Lactococcus lactis , subsp. cremoris and Lactococcus lactis , subsp. lactis , biovar. diacetylactis ) was investigated by monitoring impedance changes. The inhibitory effect of honey doses was evaluated from the course of impedance curves by determining the detection time (DT), the time of maximal difference in impedance change (IT) and the slope of the linear part of the impedance curve (K). Lactobacillus acidophilus was sensitive to the honey concentrations over 5% regardless of the heat treatment of honey. The maximal inhibitory effect on the growth of the mesophilic starter was produced by unheated honey at 10%, wt/vol, while the addition of sterilized honey had only limited influence on this culture at any concentration studied.

The role of autolysis of lactic acid bacteria in the ripening of cheese

Abstract The importance of autolysis of lactic acid bacteria in cheese ripening is evident from the literature. However, the mechanisms and the consequences still require investigation. The consequences of autolysis of mesophilic starters in Cheddar cheese are discussed and highlights from current physiological and genetic studies on starter autolysis are presented. The relative merits of measuring starter autolysis in cheese by viable starter cell densities, electron microscopic observations and assay of cell-free cytoplasmic enzymes are discussed for cheese studies using different starter strains and added phage to achieve different levels of autolysis. The balance of both the intact and autolysed starter cells in young curd appear to be important in cheese ripening. The intact cells are necessary for physiological reactions such as lactose fermentation and oxygen removal and possibly for a number of flavour reactions. In contrast, the main consequence of autolysed cells in cheese is to accelerate the peptidolytic reactions. The possible influences of autolysis of adventitious lactic acid bacteria during cheese ripening are discussed.

Generalized model of the effect of pH on lactate fermentation and citrate bioconversion in Lactococcus lactis ssp. Lactis biovar. diacetylactis

An aroma-imparting mesophilic lactic starter (Lactococcus lactis ssp. lactis biovar. diacetylactis) was studied in batch culture in medium with 50 g·l−1 lactose and 2 g·l−1 citrate. The effect of pH on the physiology of growth and the production of flavour compounds was investigated with a mathematical model. The specific rates of growth and of lactose fermentation obeyed a law of non-competitive inhibition by lactic acid produced, inhibition increasing as the pH of the medium decreased. The pH thus acted indirectly by increasing the proportion of non-dissociated lactic acid, identified as the inhibiting form of lactic acid. The generalized model, taking into account the effect of pH, was tested using fermentations at pH controlled at different values (4.5–6.5), as well as with a fermentation conducted at non-regulated pH. These simulations supported the working hypotheses. The effect of pH on the fermentation of citric acid resulted in an increase in the maximal specific rate of citrate utilization, in the bioconversion yield, and in the constant of diacetyl and acetoin reduction at acid pH. The production of flavour compounds is a complex phenomenon resulting from the interaction of pH, citric acid concentration, and the physiological state of the cells. These results are discussed with respect to the use of this strain in the preparation of manufactured dairy products.

Starters as finishers: Starter properties relevant to cheese ripening

Abstract Although the properties of starters relevant to cheese manufacture have been extensively studied and reviewed, there is little detailed information available on the contribution of starters to cheese ripening. Using extensive reference to in-vitro and in-situ studies on ripening which are currently being carried out in the authors' laboratories, this review concentrates on the roles of mesophilic starters in Cheddar cheese ripening and gives examples of data pertinent to the concepts under consideration. Particular attention is paid to the hydrolytic production of peptides, amino acids, free fatty acids and glycerol through primary ripening processes and to the production of ammonia through secondary ripening reactions. The general lack of information on the significance of starters to secondary ripening reactions important for the production of flavour compounds is highlighted. Enzymological, chemical and physiological factors involved in ripening are discussed, including concepts of starter strain variation in the activities, specificities, stabilities, concentrations and compartmentalisation of enzymes involved in ripening. The effects of the cheese environment on enzyme function and microbial physiology, including variations in the resistance of starter cells to lysis, are also discussed. The validity of using the parameter of colony forming units/g cheese (i.e. viable cell densities recovered) as a reference for comparison of levels of ripening enzyme levels is examined, as is the relationship between cell density, cell mass and enzyme concentrations.

Screening of Commercially Available Mesophilic Dairy Starter Cultures: Biochemical, Sensory, and Microbiological Properties

Sixteen commercially available mesophilic starters for the production of cultured milk were examined for sensory, biochemical, and microbiological properties. Tests were based on laboratory productions of cultured products at different fat concentrations. The diacetyl and acetaldehyde concentrations in cultured milks (2.5% fat) varied between .2 and 25.2ppm and .1 and 9.6ppm, respectively. The odor intensities of the products were mainly rated as medium and high and the flavor as mild and buttery. Textural attributes given by trained panelists were mostly fluid, creamy, and firm. Proteolysis during fermentation was generally comparable with that of yogurt starters. Cultures did not exhibit pronounced overacidification behavior during cooled storage of the products for 2 wk. The CO2-producing properties differed among the cultures. Some of the starters enriched thiamin, pyridoxine, folic acid, and pantothenic acid, whereas, on an average, the riboflavin, cobalamin, and orotic acid concentrations were reduced during fermentation. Streptococci counts in the fresh products ranged from 5.2 × 107 to 1.9 × 109 cfu/ml; corresponding counts of aroma-producing bacteria were between 8.6 × 106 and 1.7 × 109 cfu/ml.

Characterization of Leuconostoc Isolates from Commercial Mixed Strain Mesophilic Starter Cultures

In order to understand better the properties of mixed strain mesophilic starter cultures, 155 Leuconostoc mesenteroides ssp. cremoris and Leuconostoc lactis isolates were purified from three commercial cultures. Isolates were divided into groups based on plasmid content. All strains contained between 2 and 6 plasmids, ranging in size from 1 to 30 MDa One group of Leuc. lactis and five groups of Leuc. mesenteroides ssp. cremoris were obtained. The sensitivity to bacteriophage and pattern of DNA fragments when total genomic DNA was digested with the restriction enzyme EcoRI showed that strains within a plasmid group are related. Thus, plasmid analysis is as valid a technique for strain identification of Leuconostoc as it is for other dairy lactic acid bacteria. Each culture contained at least two different groups of Leuconostoc.

Detection of propionic acid bacteria in cheese.

Mesophilic lactic starters and thermophilic lactobacilli but not Streptococcus salivarius subsp. thermophilus grew on the sodium lactate agar (SLA) used for estimating the numbers of propionic acid bacteria (PAB) in cheese. The addition of cloxacillin (4 micrograms/ml) to SLA inhibited the starter bacteria but had no effect on the PAB. It was possible to count low numbers of PAB in the presence of high numbers of starter bacteria. A correlation coefficient of 0.9 was obtained between the level of propionic acid and the counts of PAB in cheese (n = 40). A disadvantage of the medium is that other bacteria found in cheese (mesophilic lactobacilli, enterococci, Clostridium tyrobutyricum) also grow on it; however, these bacteria are easily distinguishable from PAB on the basis of size, colour and absence of catalase.

In vivo genetic exchange of a functional domain from a type II A methylase between lactococcal plasmid pTR2030 and a virulent bacteriophage

The conjugative plasmid pTR2030 confers bacteriophage resistance to lactococci by two independent mechanisms, an abortive infection mechanism (Hsp+) and a restriction and modification system (R+/M+). pTR2030 transconjugants of lactococcal strains are used in the dairy industry to prolong the usefulness of mesophilic starter cultures. One bacteriophage which has emerged against a pTR2030 transconjugant is not susceptible to either of the two defense systems encoded by the plasmid. Phage nck202.50 (phi 50) is completely resistant to restriction by pTR2030. A region of homology between pTR2030 and phi 50 was subcloned, physically mapped, and sequenced. A region of 1,273 bp was identical in both plasmid and phage, suggesting that the fragment had recently been transferred between the two genomes. Sequence analysis confirmed that the transferred region encoded greater than 55% of the amino domain of the structural gene for a type II methylase designated LlaI. The LlaI gene is 1,869 bp in length and shows organizational similarities to the type II A methylase FokI. In addition to the amino domain, upstream sequences, possibly containing the expression signals, were present on the phage genome. The phage phi 50 fragment containing the methylase amino domain, designated LlaPI, when cloned onto the shuttle vector pSA3 was capable of modifying another phage genome in trans. This is the first report of the genetic exchange between a bacterium and a phage which confers a selective advantage on the phage. Definition of the LlaI system on pTR2030 provides the first evidence that type II systems contribute to restriction and modification phenotypes during host-dependent replication of phages in lactococci.

Growth and Activity of Mesophilic Lactic Acid Streptococci in Ultrafiltered Skim Milk and in Reconstituted Nonfat Dry Milk of Differing Total Solids Contents

The activity of mesophilic starter cultures Streptococcus lactis C2, Streptococcus cremoris SK11, and Streptococcus diacetylactis DRC3 in 8.3 and 15% total solids reconstituted skim milk was compared with growth of these organisms in 8.9% total solids skim milk (unconcentrated control) and in UF concentrated skim milk of 12 and 13% total solids. The starter cultures were inoculated into the different milk samples and incubated at 30°C for 15 to 16h. Samples were taken periodically for determination of pH, percentage titratable acidity, and cell populations. Cultures grown in UF concentrated milk consistently exhibited higher cell numbers and developed greater acidity than when grown in reconstituted skim milk of similar solids content. Streptococcus cremoris SK11 was the most active culture in acid development in the various media. Growth was not related to solids level or milk type, that is, retentate or reconstituted skim milk. A model for predicting developed acidity from solids content and from type of milk was determined by multiple regression analysis. The results suggest that UF concentrated skim milk is superior to reconstituted skim milk as a substrate for lactic acid bacteria and that skim milk retentates have potential for use as media for starter cultures.

The use of mesophilic cultures in the dairy industry.

The use of mesophilic starter cultures, containing group N Streptococcus and Leuconostoc species, in the dairy industry is examined. Bacteriophage attack is identified as the main cause of culture inhibition and criteria used to select stable mixed-strain starter cultures and phage-insensitive defined strains are established. The key aspects of culture propagation and storage are highlighted, as well as bulk-culture protection based on physical exclusion of phage and the use of phage-inhibitory media. Developments in the application of starter culture systems in different countries are examined and show that significant process control and elimination of phage problems can be achieved.