Technological characterization of lactic acid bacteria isolated from Armada cheese (a Spanish goats’ milk cheese)

Thirty-one strains of lactic acid bacteria (LAB) isolated from Armada cheese, Sobado variety, (eight strains of Lactococcus lactis subsp. lactis, four strains of Lactococcus lactis subsp. cremoris, two strains of L. lactis subsp. lactis biovar. diacetylactis, two strains of Leuconostoc mesenteroides subsp. mesenteroides, two strains of Leuconostoc mesenteroides subsp. dextranicum, five strains of Lactobacillus plantarum, six strains of Lactobacillus casei subsp. casei and two strains of Lactobacillus brevis) were screened for their acidifying capacity and enzymatic activity, that included the rapid API-ZYM system, the proteolytic activity, the amino-, di-, and carboxypeptidase activity and the caseinolytic activity. The strains of L. lactis subsp. lactis exhibited the highest acidifying and proteolytic activity. Lipase and esterase activity was practically non-existent for lactococci and lactobacilli; a certain esterase activity was observed among leuconostoc. The highest aminopeptidase activity was demonstrated by the cell-free extract (CFE) of some strains of L. plantarum, L. casei subsp. casei and L. mesenteroides subsp. dextranicum. The CFEs of L. lactis subsp. cremoris and L. lactis subsp. lactis possessed carboxypeptidase and dipeptidase activities, at levels depending on the strain. Appreciable caseinolytic activity was detected for the CFE of L. plantarum and those some lactococci.     

Peptidase and proteinase activity ofLactococcus lactis,Lactobacillus casei andLactobacillus plantarum

The proteolytic system of several non-commercial strains of lactococci and lactobacilli that were isolated directly from traditional-Spanish, semi-hard, goats' milk cheese was studied. The aminopeptidase, X-prolyldipeptidyl aminopeptidase, dipeptidase and proteinase activity of these new strains was measured for the cytoplasmic, cell-wall/membrane and spontaneously released fractions. The aminopeptidase activity was exclusively intracellular and higher forLactobacillus casei subsp.casei than forLactococcus lactis subsp.lactis. Lactobacillus plantarum showed higher dipeptidase activity thanL. casei. The highest level of proteinase activity was recorded for the cell-wallmembrane fraction ofLactococcus lactis subsp.lactis IFPL 359, and was higher on β-casein than on α_s-casein for all the strains studied. These results suggest some different contribution of these strains to the proteolysis of cheese during ripening and they seem to complement each other when used together in the starter culture.     

Peptide hydrolase system of lactic acid bacteria isolated from Serra da Estrela cheese

Lactobacillus paracasei subsp. paracasei ESB 230, Leuconostoc mesenteroides subsp. mesenteroides ESB 136, Lactococcus lactis subsp. lactis ESB 117 and Enterococcus faecium ESB 50, previously isolated from certified Serra da Estrela cheeses, were tested for their aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, dipeptidase and carboxypeptidase activities. The crude cell-free extracts (CFE) of Lb. paracasei ESB 230 exhibited the highest aminopeptidase activity, followed by CFE of Leuc. mesenteroides ESB 136 and, at last, by CFE of L. lactis ESB 117; the aminopeptidase activity in CFE of Ent. faecium was practically non-existent. The four CFE studied also showed appreciable carboxypeptidase activities, although these were lower than their dipeptidase counterparts; in addition, their dipeptidyl aminopeptidase and endopeptidase activities were lower than their aminopeptidase activities. Dipeptides consisting of hydrophobic amino acid residues (i.e. leucine, methionine and phenylalanine) were more rapidly attacked by all CFE than those with hydrophilic amino acid residues. The peptide hydrolase system of CFE of Lb. paracasei ESB 230 was qualitatively quite similar to, but quantitatively more active than that of CFE of Leuc. mesenteroides ESB 136 (except for the endopeptidase); additionally, the CFE of L. lactis ESB 117 and of Ent. faecium ESB 50 were quite distinct from each other, and from the other two CFE tested.     

Enzymatic activity of lactic acid bacteria (with antimicrobial properties) isolated from a traditional Spanish cheese

Twenty-four strains of lactic acid bacteria (LAB) isolated from a traditional Spanish cheese (Genestoso cheese) were evaluated for their enzymatic activities (acidifying and proteolytic abilities and carboxypeptidase, aminopeptidase, dipeptidase, caseinolytic and esterase activities), in order to select indigenous strains of technical interest for the manufacture of cheese. These strains were selected on the basis of their antimicrobial activity relative to five reference strains and were identified as Lactococcus lactis subsp. lactis (thirteen strains), Leuconostoc mesenteroides (two strains), Leuconostoc pseudomesenteroides (one strain), Lactobacillus paracasei (two strains), Lactobacillus plantarum (one strain) and Enterococcus faecalis (five strains).     

Intragenomic 16S rRNA gene heterogeneity in Lactococcus lactis subsp. cremoris

Partial 16S rRNA gene sequencing of Lactococcus lactis subsp. cremoris strains from our collection identified strains containing sequences diagnostic of both subspecies L. lactis subsp. lactis and L. lactis subsp. cremoris together in the same strain. The presence of a plasmid-encoded 16S rRNA pseudogene partly explained this result. Twenty-four out of 46 L. lactis subsp. cremoris strains tested by PCR contained this pseudogene. However, further analysis showed that five of these 24 strains also contained chromosomal 16S rRNA genes with sequences typical of L. lactis subsp. lactis. Genetic and phenotypic tests indicated these strains were otherwise normal L. lactis subsp. cremoris strains. Past recombination events between the 16S rRNA pseudogene and chromosomal 16S rRNA genes may explain this phenomenon. Genomic heterogeneity for both 16S rRNA and other gene sequences was observed for L. lactis subsp. cremoris SK11 from different laboratories, indicating caution is needed when integrating data from diverse sources for nominally the same strain.     

Determination of some characteristics coccoid forms of lactic acid bacteria isolated from Turkish kefirs with natural probiotic

In this study, a total of 21 coccoid forms of lactic acid bacteria (lactococci) were isolated from Turkish kefir samples. As a result of the identification tests, 21 lactococci isolates were identified as Lactococcus cremoris (11 strains), Lactococcus lactis (4 strains), Streptococcus thermophilus (3 strains) and Streptococcus durans (3 strains). The amount of produced lactic acid, hydrogen peroxide, proteolytic activity, diacetyl and acetaldehyde productions of the lactococci were determined. Different amounts of lactic acid were produced by strains studies; however, lactic acid levels were 2.3-9.9 mg/ml. While Lac. lactis Z13S, Str. durans Z7S, Z8S, Z15S, Lac. cremoris Z9S, Z16S, Z17S, Z19S, Z20S and Z21S strains were not shown hydrogen peroxide, Lac. lactis Z1S and Z2S strains had a maximum hydrogen peroxide (0.17 μg/ml). Lac. lactis Z2S, Z3S, Str. thermophilus Z5S, Z12S, Str. durans Z7S, Z8S, Lac. cremoris Z14S and Z16S strains were not show proteolytic activity, Lac. cremoris Z20S strain produced the maximum amount (0.09 mg/ml) of proteolytic activity. Acetaldehyde concentration produced in Lactobacillus strains ranged between 0.18 and 3.96 μg/ml. Antimicrobial effects of the lactococci on Escherichia coli NRLL B-704, Staphylococcus aureus 4-63, Pseudomonas aeruginosa ATCC 29212 were also determined by an agar diffusion method. All of the strains were able to inhibit S. aureus, while Lac. lactis Z1S, Z2S and Lac. cremoris Z6S strains were able to inhibit E. coli and P. aeruginosa. Also, Str. thermophilus Z5S strain were able to inhibit P. aeruginosa.     

Efficacy of four conjugal lactococcal phage resistance plasmids against phage in commercial Lactococcus lactis subsp. cremoris cheese starter strains

The efficacy of four lactococcal phage resistance plasmids (pNP40, pMU1311, pDI60 and pKP100) against phage was assessed after their conjugal transfer to four commercial Lactococcus lactis subsp. cremoris cheese starter strains and to the plasmid-free strain L. lactis subsp. cremoris MG1363. In MG1363, only pNP40 conferred resistance to prolate phages c2 and 643. Highest levels of resistance to small isometric phages in MG1363 occurred when pNP40 was stacked together with pMU1311 or pDI60. In the four starter strains, the plasmids conferred varying levels of resistance to small isometric phages. Growth and acidification rates in milk of most transconjugants derived from the starter strains decreased, but this was not always due to loss of plasmid-encoded cell wall proteinase (lactocepin) activity. Only one transconjugant grew during repeated subculture in milk with addition of factory wheys containing phages. This and the presence of bacteriocins encoded on pMU1311 and pDI60 limited application of the plasmids to protect L. lactis subsp. cremoris starters against phages in industry. However, some of the plasmids could be useful in extending the industry life of starters where fast acid production is not required or where bacteriocin production is acceptable.     

Differentiation of intracellular peptidases of starter and adjunct cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Selection of starter and adjunct cultures is important to minimize bitterness of Cheddar and Gouda cheeses. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry may be useful for rapid screening of cheese cultures for propensity to produce bitter cheese. The objective of this study was to demonstrate the application of MALDI-TOF for differentiating intracellular peptidase activities of starter and adjunct cultures on β-CN f193-209 under simulated cheese condition. Bovine β-casein was incubated with chymosin in 9.55 g/l citrate buffer (pH 5.4, 40 g/l sodium chloride) at 30°C for 24 h, followed by incubation with cell-free extract (CFE) of starter or adjunct culture. Mixed strains of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris designated as 56 and 105 were the sources of nonbitter and bitter starter cultures, respectively. Lactobacillus helveticus WSU-19 and W900R represented adjunct cultures having high and low debittering activities, respectively. The degradation pattern of β-CN f193-209 by CFE of WSU-19 indicates general aminopeptidase and endopeptidase activities, while degradation of the peptide by CFE of W900R, 56, and 105 are mainly from endopeptidase activity. The rates of β-CN f193-209 hydrolysis by CFE of WSU-19, W900R, 56, and 105 are 6.90, 0.38, 0.39, and 0.23 mg/l per h, respectively.     

Purification and characterization of a dipeptidase from Lactobacillus curvatus DPC2024

A dipeptidase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-sephacel, phenyl sepharose, chelating sepharose fast flow and MonoQ. The purified dipeptidase was a monomer of ∼52 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. The enzyme was optimally active at pH 8 and 50°C and retained ∼10% of its maximum activity after pre-heating for 10 min at 70°C. The enzyme was a metallopeptidase, strongly inhibited by 0.1 mM ethylenediaminetetraacetic acid and o-phenanthroline and reactivated by a number of divalent metal ions. The enzyme was also inhibited by p-chloromercuribenzoate and β-mercaptoethanol. The enzyme was a strict dipeptidase, capable of hydrolysing a range of dipeptides but not tri-, tetra- or pentapeptides, p-nitroanilide derivatives of amino acids nor N- and C-terminal-blocked dipeptides. The N-terminal amino acid sequence of the first 20 residues showed significant homology with dipeptidases from Lactobacillus delbrueckii subsp. lactis DSM 7290 and Lactococcus lactis subsp. cremoris MG1363.     

Protease and Peptidase Activity of Micrococcus Species

Nine different strains or species of micrococci were examined for intracellular protease and peptidase activity. Highest activities of leucine, alanine, and methionine aminopeptidases and proline iminopeptidase were found in Micrococcus sp. ATCC 398. Lysine aminopeptidase and dipeptidase activities were maximum in Micrococcus freudenreichii ATCC 407. Highest intracellular protease activity appeared in Micrococcus sp. LL3 isolated from Cheddar cheese. All strains except Micrococcus sp. ATCC 398 preferentially hydrolyzed the β-casein component during growth in skim milk at 30°C, and after longer incubation whole casein (α_s1 and β) was completely hydrolyzed. Micrococcus sp. ATCC 398 failed to grow in skim milk under our experimental conditions. This pattern of proteolysis also was observed when skim milk was incubated with cell-free extracts of all micrococci tested as well as during growth of Micrococcus caseolyticus ATCC 13548 in ultrafiltered milk (five-fold concentration).     

Influence of proteolytic Lactococcus lactis subsp. cremoris on ripening of reduced-fat Cheddar cheese made with camel chymosin

This study investigated proteolysis in reduced-fat Cheddar cheese produced with camel chymosin and Lactococcus lactis subsp. cremoris with the ability to cleave the N-terminus of α_S1-casein. The aim was to match the activity of bovine chymosin, which leads to softer cheese structure than camel chymosin. Cheeses were analysed for gross composition, casein and peptide breakdown, release of free amino acids, structure parameters and sensory characteristics. Selected Lc. lactis subsp. cremoris increased the amount of peptides and, to a limited extent, the total amount of free amino acids in the cheeses. One group of experimental cheeses was found to have a significantly firmer structure, higher stress at fracture and modulus of deformability than the reference cheeses. The addition of the selected proteolytic dairy strains of Lc. lactis subsp. cremoris to the cheeses did not result in extended breakdown of α_S1-casein or a softer cheese structure.     

The effects of freezing and frozen storage of ewe's milk cheese on the viability and proteolytic activity of lactococci used as a starter

A study has been conducted on the effect of two freezing conditions (slow and fast) and frozen storage of ewe's milk cheese (?20 °C for 4 months) on the viability and the proteolytic (cascinolytic and aminopeptidase) activity ofLactococcus lactis subsp.lactis andL. lactis subsp.cremoris used as starters in the manufacture of cheese. The study was carried out on the lactococci subjected to freezing and frozen storage, either in the cheeses or in curds simulating a model system. As well as other parameters used to quantity microbial activity, the total viable counts during the subsequent cheese ripening have been analysed in the frozen cheeses. Frozen storage of the investigated lactococci, either in the cheese or in model systems, gave rise to significant decreases in the caseinolytic and aminopeptidase activities greater than did freezing alone. No clear differences were found in enzymatic activity values when using slow or fast freezing conditions. Storage of the cheeses under frozen conditions also affected microbial viability and consequently caused a greater decrease of the viable flora during subsequent ripening of the frozen stored cheeses.     

Protease and esterase activity of staphylococci

The aim of this work was to characterize protease and esterase activities of staphylococci in order to establish if they could contribute to the release of amino acids and short-chain fatty acids during ripening of fermented sausages. Eighteen Staphylococcus strains belonging to the species Staphylococcus xylosus (5), S. saprophyticus (3), S. equorum (4), S. carnosus (4) and S. simulans (2), previously isolated from different types of Southern Italian fermented sausages, were screened for proteinase, aminopeptidase and esterase activities. Most of the staphylococci strains lacked detectable levels of proteinase activity against casein-fluorescein isothiocynate. In the active strains, this activity was extracellular or cell-envelope associated. The studied staphylococci strains also showed low levels of aminopeptidase activities, which preferentially hydrolysed substrates containing L-methionine, L-leucine and L-phenylalanine as N-terminal residue. In contrast, all staphylococcal strains possessed significant activity against short-chain fatty acid esters. The maximum esterase activities were detected in whole-cell suspensions and cell-free extracts and to a lesser extent in the extracellular medium. The substrates preferentially hydrolysed were rho-nitrophenyl (rho-NP) butyrate and rho-NP caprylate and, secondly, rho-NP palmitate. The extracellular extracts of most of the strains were only active against rho-NP butyrate except for those of S. equorum (SI3, SI4) and S. simulans (Ssm12, Ssm21), which also hydrolysed rho-NP caprylate and rho-NP palmitate. The cell-free extracts and whole cells were mainly active against rho-NP butyrate and rho-NP caprylate, showing activity levels from 1760 U/mg of proteins to 54 U/mg of proteins and from 12,200 U/mg of proteins to 133 U/mg of proteins respectively. These activities were especially high in the strains that belonged to S. xylosus and S. equorum species. The diversity of the studied metabolic properties and, especially, the esterase activities in different staphylococcal species and even strains of the same species emphasize the relevance of these in vitro characterization studies for a rational selection of new starter cultures.     

Gelation and resistance to shearing of fermented milk: Role of exopolysaccharides

Milk was fermented with the exopolysaccharide-producing (EPS⁺) strains Lactococcus lactis subsp. cremoris, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and with the non-EPS-producing strain (EPS⁻) L. lactis subsp. cremoris. The kinetics of gelation and the behaviour of set fermented milk during and after shearing were studied using rheometry and confocal scanning laser microscopy. The time of gelation of milk depended on the kinetics of acidification of strains whereas the pH of gelation depended mostly on the presence of exopolysaccharides (EPS). In set fermented milk with EPS⁺ strains, bacteria were observed in protein-free areas likely filled with EPS. Phase-separated EPS and caseins contributed to induce the gelation of fermented milk at pH 5.6. The high resistance to shearing of milk fermented with the EPS⁺ strain L. lactis subsp. cremoris might be due to the negative charge of the exopolysaccharide allowing an attractive interaction with caseins.     

Stability of autolytic lactococci during starter production and storage in commercial whey-based media

Lactococcus lactis ssp. cremoris strains with different autolytic activities were grown in milk and in a commercial whey-based medium. Fermentation under pH control substantially improved total and viable counts of strain NM33-7; highest populations were obtained on the commercial whey-based medium and the cultures had the highest content of viable cells (80%). Cultures grown in milk without pH control gave only 46% viable cells. Once fermentation was completed, cooling prevented a significant decrease in viable counts. Losses in viability of NM33-7 upon extended incubation at 30 °C or storage at 4 °C were higher when the cultures were prepared on whey-based medium than when prepared on milk without pH control. In pH-controlled fermentations, the autolytic activity of NM33-7 was higher when grown on whey-based medium than when grown on milk. A negative correlation was obtained between the autolytic activity of the Lc. lactis ssp. cremoris strains and their subsequent stability during storage.     

Comparison of growth and survival of single strains of Lactococcus lactis and Lactococcus cremoris during Cheddar cheese manufacture

Traditionally, starter cultures for Cheddar cheese are combinations of Lactococcus lactis and Lactococcus cremoris. Our goal was to compare growth and survival of individual strains during cheesemaking, and after salting and pressing. Cultures used were 2 strains of L. lactis (SSM 7605, SSM 7436) and 2 strains of L. cremoris (SSM 7136, SSM 7661). A standardized Cheddar cheese make procedure was used that included a 38°C cook temperature and salting levels of 2.0, 2.4, 2.8, 3.2, and 3.6% from which were selected cheeses with salt-in-moisture levels of 3.5, 4.5, and 5.5%. Vats of cheese were made using each strain on its own as biological duplicates on different days. Starter culture numbers were enumerated by plate counting during cheesemaking and after 6 d storage at 6°C. Flow cytometry with fluorescent staining by SYBR Green and propidium iodide was used to determine the number of live and dead cells in cheese at the different salt levels. Differences in cheese make times were strain dependent rather than species dependent. Even with correction for average culture chain length, cheeses made using L. lactis strains contained ～4 times (～0.6 log) more bacterial cells than those made using L. cremoris strains. Growth of the strains used in this study was not influenced by the amount of salt added to the curd. The higher pH of cheeses with higher salting levels was attributed to those cheeses having a lower moisture content. Based on flow cytometry, ～5% of the total starter culture cells in the cheese were dead after 6 d of storage. Another 3 to 19% of the cells were designated as being live, but semipermeable, with L. cremoris strains having the higher number of semipermeable cells.     

Lysis of starters in UF cheeses: Behaviour of mesophilic lactococci and thermophilic lactobacilli

The objective of this work was to study the autolytic behaviour of strains of mesophilic (Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris) and thermophilic lactic acid bacteria (Lactobacillus helveticus, Lb. delbrueckii subsp. lactis and Streptococcus thermophilus) in UF cheese. Cheeses were made from a UF-retentate (milk concentrated by a factor of 6) of microfiltered milk (0.8 μm pore size membrane) using the following starter systems: (1) single inocula of autolytic strains of L. lactis (US3, AM2 or AM1), non-autolytic strains of L. lactis (AM2-C or CNRZ-144), (2) a co-inocula of strains of Lb. helveticus (ITG-LH1, CNRZ-32 or CNRZ-303), Lb. delbrueckii subsp. lactis (ITG-LL14 or ITG-LL51) with the same strain of S. thermophilus CNRZ-1358. Cell viability was monitored over a 28 day ripening period by enumeration on selective media. Degree of lysis was determined by the measurement of the intracellular marker lactate dehydrogenase (LDH) activity, and also by immunodetection of intracellular proteins with species specific antibodies. In UF cheeses, lysis of autolytic strains of L. lactis was significantly delayed, showing release of intracellular components after 21 days of ripening. No lysis was observed for non-autolytic L. lactis strains or for S. thermophilus. Lysis of thermophilic lactobacilli (Lb. helveticus, Lb. delbrueckii), was observed from the start of ripening, but the onset and the level of lysis observed was strain and species dependent.     

Sensitivity of Commercial Cheddar Cheese Starter Isolates to Bacteriophage Associated with Wheys

Seventy-six Cheddar cheese starter cultures were collected from five cheese plants located in New York, Wisconsin, and Illinois. Several single colony isolates from each starter culture were purified and identified to species on the basis of six biochemical reactions. Thirteen of the 76 starter cultures were identified as mixture of Strepcococcus lactis and S. cremoris and 63 of the starter cultures were single species. Plasmid profiles and resistance to five phages (ML3, C2, TRM, 18-16, and C5W6) were determined for all 450 isolates. One hundred and thirty-six isolates that constituted a representative cross-section of commercial strains were selected upon their plasmid and phage-resistance profiles. These strains were used as indicators to test phage activities of 57 wheys collected from four of the five cheese plants. Wheys from different cheese plants showed varying levels of phage activity. Wheys from the plant that had the most acute phage problem completely lysed at least 25% of the 136 strains. Most of the sensitive bacterial strains of the 136 isolates were lysed by wheys from two individual plants. The percentage of strains from each plant resistant to all 57 wheys ranged from 9 to 43.     

Peptidase and esterase activities from mutant strains of Lactobacillus casei

Cell-free extracts from four Lactose-negative strains of Lactobacillus casei and their parents were evaluated for peptidase and esterase activity. The four strains showed aminopeptidase (AP), dipeptidase (DP), carboxypeptidase (CP), aryl-peptidyl amidase (APA) and caseinolytic activities. Very little differences can be noticed between the AP and DP levels of the different strains. On the other hand there were significantly higher CP activities in the Lac(+) strains 64H and ATCC 4646 when compared to the Lac(-) derivatives. A difference in activity between the Lac(+) and its Lac(-) derivative was also observed for the APA and caseinolytic activities. Parent and mutant strains of Lactobacillus casei posessed three active AP bands, with RF values of 0.18, 0.21 and 0.31 and one DP band with RF value of 0.25. An additional band (0.32) was detected in strain Lactobacillus casei ATCC 4646 Lac(+) and its mutant. As a general rule the rate of hydrolysis of the p-nitrophenyl esters of fatty acids for the parent and mutant strains of Lactobacillus casei was higer than that of the o-nitrophenyl derivatives. All the strains tested exhibited an esterase band with an RF value of 0.3, while only one substrate was hydroysed by the band showing a lower RF value. α-Naphthyl butyrate was the only substrate hydrolysed by this enzyme in the case of Lactobacillus casei 64H and 11578 while it was α-naphthyl acetate for ATCC 4646.     

Screening for differences in the proteolytic systems of Lactococcus, Lactobacillus and Propionibacterium

The caseinolytic, and the endo- and aminopeptidase activities of the intracellular (IC) and cell-envelope-associated (CE) fractions of selected strains of Lactococcus, Lactobacillus and Propionibacterium have been compared. With the exception of one Lactococcus strain, most of the caseinolytic activity of the three genera was located in the IC fraction, as was the case for the amino- and endopeptidases. The lactococci showed low activity on Pro-pNA and high activity on Gly-Pro-pNA, the reverse was characteristic for the propionibacteria (PAB) while lactobacilli took an intermediary position. Lactococcus lactis ssp. cremoris INF-C12 was the strain with the highest total endopeptidase activity. The experiment with phosphoramidon and a peptide inhibitor, β-casein f58–72 (β-CN f58–72), indicated differences of IC endopeptidases of lactic acid bacteria (LAB) and PAB. In contrast to the LAB endopeptidases, the PAB endopeptidases were little inhibited by β-CN f58–72, and were not affected by phosphoramidon. Lysozyme was used to produce spheroplasts from whole cells; however, the susceptibility to such treatment varied. Intracellular material, ≥97%, was easily released from the lactococci strains, for other strains additional sonication was most often necessary for ≥93% lactate dehydrogenase release.