Kinetics of the thermal inactivation of the Lactococcus lactis bacteriophage P008

The thermal resistance of the lactococcal bacteriophage P008 was investigated between 55 and 80 degrees C. Inactivation kinetics revealed an order of reaction above 1 and could be determined by a non-1st-order regression model. Phage inactivation was influenced by the medium (milk and Ca-M17-broth). Within the investigated temperature range, milk had a protective effect on phage P008. This was reflected in the rate constant and in the activation energy. Thermal phage inactivation studies reported in literature were re-analysed using non-1st-order regression. The obtained kinetic parameters showed that phage P008 belongs to the most heat resistant lactococcal phages investigated so far.     

Interactions between Lactococcus lactis and Streptococcus thermophilus strains in Cheddar cheese processing conditions

The growth of pure and mixed cultures of Lactococcus lactis and Streptococcus thermophilus under simulated Cheddar cheese manufacture was examined. Cell-free wheys (CFW) of the cultures were prepared for analysis by automated spectrophotometry (AS). The maximal growth rate of the lactococci in S. thermophilus R0083 CFW was 13% higher than that noted in their own CFW and three lactococci also gave higher biomass levels (OD_max). During simulated Cheddar cheese fermentations with four paired cultures, one L. lactis strain grew 20% less when paired with S. thermophilus R0083, and an increase in colony forming units (cfu) was found with one other lactococcal strain. Viable counts of S. thermophilus in mixed cultures varied by less than 0.1 log cfu mL^?1. The AS data on OD_max in CFW were useful in predicting the evolution of cfu in the fermented mixed cultures. As a function of strain, the presence of S. thermophilus in a Cheddar fermentation process can enable extended growth of the lactococci.     

Proteolysis of Hispanico cheese manufactured using lacticin 481-producing Lactococcus lactis ssp. lactis INIA 639

Hispánico cheese was manufactured using lacticin 481-producing Lactococcus lactis ssp. lactis INIA 639, bacteriocin-nonproducing L. lactis ssp. lactis INIA 437, or a combination of both strains, as starter cultures. Lactobacillus helveticus LH 92, a culture of high amino-peptidase activity sensitive to lacticin 481, was added to all vats. Milk inoculation with the bacteriocin producer promoted early lysis of Lb. helveticus cells in cheese. Cell-free aminopeptidase activity in cheese made with the 3 lactic cultures was 1.8 times the level reached in cheese made only with L. lactis strain INIA 437 and Lb. helveticus, after 15 d of ripening. Proteolysis (as estimated by the o-phthaldialdehyde method) in cheese made with the 3 lactic cultures was twice as high, and the level of total free amino acids 2.4 times the level found in cheese made only with L. lactis strain INIA 437 and Lb. helveticus, after 25 d of ripening. Hydrophobic and hydrophilic peptides and their ratio were at the lowest levels in cheese made with the 3 lactic cultures, which received the lowest scores for bitterness and the highest scores for taste quality.     

Thermal inactivation of chymosin during cheese manufacture

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100 +/- 21 min and a z-value of 5.9 +/- 0.3 degrees C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.     

Formation of methional by Lactococcus lactis IFPL730 under cheese model conditions

The present work describes the capacity of Lactococcus lactis to produce methional and other sulphur compounds derived from methionine (Met) in a cheese model system. Cheese slurries were prepared from pasteurized ewes' skimmed milk, chemically acidified with glucono-'-lactone and homogenized aseptically adding Met, !-ketoglutarate, pyridoxal 5'-phosphate, thiamine pyrophosphate and NaCl. Slurries were incubated at 12 °C for 14 days with cellular suspensions of L. lactis IFPL359, L. lactis IFPL730 and L. lactis NCDO763 in different combinations. Slurries added with resting cells and the intracellular fraction from L. lactis IFPL730 showed the highest production of methional at the outset of incubation, which decreased during incubation along with a concomitant increase in 3-methylthiopropanol. The sensorial analysis of slurries indicated a characteristic methional aroma (cooked potato-like) in samples containing 4-methylthio-2-ketobutyrate and the intracellular fraction from L. lactis IFPL730. As incubation proceeded, the intensity of methional aroma decreased but samples were judged by the panel tasters as developing a cheese-like flavour.     

Effectiveness of thermal treatments and biocides in the inactivation of Argentinian Lactococcus lactis phages

The thermal and chemical resistance levels of four autochthonal bacteriophages of Lactococcus lactis subsp. lactis, isolated from cheese processes, was investigated. The times required to obtain 99% inactivation of phages (T99) at 63 and 72 degrees C in three suspension media (M17 broth, reconstituted commercial nonfat skim milk, and Tris magnesium gelatin buffer) were determined. Thermal resistance was dependent on the phage studied, and the results of this study demonstrate that pasteurization treatments used in dairy industries may leave viable viral particles in milk. It was possible to determine that M17 broth was generally the least protective medium, while phosphate buffer was the most protective one. Peracetic acid (0.15%, vol/vol) was the most effective viricidal agent, with exposures of 5 min being sufficient to inactivate high-titer phage suspensions (>10(6) PFU/ml). To achieve total inactivation (<10 PFU/ml) of viral suspensions, sodium hypochlorite was effective at 100 ppm for only two phages, while the other two phages needed concentrations of 200 and 300 ppm. Ethanol at concentrations of 100 and 75% proved to be very efficient in inactivating phages, but isopropanol was not effective against them.     

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.     

Data mining and fuzzy modelling of high pressure inactivation pathways of Lactococcus lactis

A mathematical model to predict lethal and sublethal pressure effects on Lactococcus lactic was established through combined use of Principal Component Analysis (PCA) and Fuzzy Logic. The model was based on data comprising pressure inactivation kinetics with 64 combinations of the parameters pressure, temperature, pH, and buffer composition, samples were analysed with respect to 5 physiological states describing lethal or sublethal injury of L. lactis, i.e. viable cell counts (CFU), undamaged cell counts (CFUsub), membrane integrity (MI), metabolic activity (MA) and the activity of the membrane bound enzyme LmrP (LmrP).Correlations found by PCA were used to generate a bi-layer fuzzy model using clustering methods and rule oriented statistical analysis as well as the physiological states CFU and LmrP as autonomous output variables. The result of these variables is used to accurately predict dependent output variables MA, CFUsub, and MI taking into account the combined effects of the inactivation process.Industrial relevanceThe study provides a predictive model for the inactivation of an industrially relevant micro-organism. Predictive models are useful for process design on the industrial scale. Further, our model provides information about intermediate steps of high pressure inactivation.     

Thermal and chemical inactivation of Lactobacillus virulent bacteriophage

The effect of thermal treatments and several biocides on the viability of Lactobacillus virulent phage P1 was evaluated. Times to achieve 99% inactivation (T₉₉) of phage at different treatment conditions were calculated. The thermal treatments applied were 63, 72, and 90°C in 3 suspension media (de Man, Rogosa, Sharpe broth, reconstituted skim milk, and Tris magnesium gelatin buffer). Phage P1 was completely inactivated in 5 and 10 min at 90 and 72°C, respectively; however, reconstituted skim milk provided better thermal protection at 63°C. When phage P1 was treated with various biocides, 800 mg/L of sodium hypochlorite was required for total inactivation (～7.3 log reduction) within 60 min, whereas treatment with 100% ethanol resulted in only a ～4.7 log reduction, and 100% isopropanol resulted in a 5.2-log reduction. Peracetic acid (peroxyacetic acid) at the highest concentration used (0.45%) resulted in only a ～4.-log reduction of phage within 60 min. The results of this study provide additional information on effective treatments for the eradication of potential phage infections in dairy plants.     

High-pressure processing of Turkish white cheese for microbial inactivation

High-pressure processing (HPP) of Turkish white cheese and reduction of Listeria monocytogenes, total Enterobacteriaceae, total aerobic mesophilic bacteria, total molds and yeasts, total Lactococcus spp., and total Lactobacillus spp. were investigated. Cheese samples were produced from raw milk and pasteurized milk and were inoculated with L. monocytogenes after brining. Both inoculated (ca. 10(7) to 10(8) CFU/g) and noninoculated samples were subjected to HPP in a high-pressure food processor at 50 to 600 MPa for 5 and 10 min at 25 degrees C. Reductions in L. monocytogenes, total aerobic mesophilic bacteria, Lactococcus spp., and Lactobacillus spp. in both pasteurized- and raw-milk cheese samples and reductions in total molds and yeasts and total Enterobacteriaceae counts in raw-milk cheese samples increased with increased pressure (P < or = 0.05). The maximum reduction of the L. monocytogenes count, ca. 4.9 log CFU/g, was obtained at 600 MPa. Because of the highly inhibitory effect of pasteurization, the total molds and yeasts and total Enterobacteriaceae counts for the cheese samples produced from pasteurized milk were below the detection limit both before and after HPP. There was no significant difference in inactivation of L. monocytogenes, total aerobic mesophilic bacteria, Lactococcus spp., and Lactobacillus spp. under the same treatment conditions for the raw milk and pasteurized milk cheeses and for 5- and 10-min treatment times (P > 0.05). No significant change was detected in pH or water activity of the samples before and after HPP. Our findings suggest that HPP can be used effectively to reduce the microbial load in Turkish white cheese.     

Inhibition of Clostridium tyrobutyricum in Vidiago cheese by Lactococcus lactis ssp. lactis IPLA 729, a nisin Z producer

Lactococcus lactis ssp. lactis IPLA 729 is a nisin Z producer isolated from raw milk cheese able to grow and produce nisin Z in milk. The ability of this strain to inhibit the growth of Clostridium tyrobutyricum CECT 4011, a late blowing agent, in Vidiago cheese, a semi-hard farmhouse variety, manufactured in Asturias, Northern Spain, was investigated. For control purposes, cheeses were manufactured with the mesophilic mixed starter IPLA-001. In experimental cheeses, the nisin-producing strain L. lactis IPLA 729 was combined with this starter. Nisin Z activity reached a concentration of 1600 AU/ml in 1-day cheeses and this level was maintained until 15 days of ripening. Furthermore, to compare the inhibitory activity of the nisin-producing strain to nitrate, cheeses were also manufactured with a commercial starter culture and potassium nitrate as anti-blowing agent was added in accordance with Vidiago's cheesemakers. The control, experimental and commercial cheeses were contaminated with C. tyrobutyricum CECT 4011. The composition of the three different cheeses showed only slight differences with respect to total solids, protein and fat, although control and experimental cheeses showed a richer flavour-compound profile than commercial cheeses. The level of the spoilage strain C. tyrobutyricum CECT 4011 decreased from 1.2x10(6) to 1.3x10(3) cfu/g during ripening in presence of the nisin Z producer, while it increased to 1.99x10(9) cfu/g in control cheeses and to 3.5x10(7) cfu/g in commercial cheeses.     

Enhanced nutty flavor formation in cheddar cheese made with a malty Lactococcus lactis adjunct culture

Nutty flavor in Cheddar cheese is desirable, and recent research demonstrated that 2- and 3-methyl butanal and 2-methyl propanal were primary sources of nutty flavors in Cheddar. Because malty strains of Lac-tococcus lactis (formerly Streptococcus lactis var. malti-genes) are characterized by the efficient production of these and other Strecker aldehydes during growth, this study investigated the influence of a malty L. lactis adjunct culture on nutty flavor development in Cheddar cheese. Cheeses made with different adjunct levels (0, 10⁴ cfu/mL, and 10⁵ cfu/mL) were ripened at 5 or 13°C and analyzed after 1 wk, 4 mo, and 8 mo by a combination of instrumental and sensory methods to characterize nutty flavor development. Cheeses ripened at 13°C developed aged flavors (brothy, sulfur, and nutty fla-vors) more rapidly than cheeses held at 5°C. Additionally, cheeses made with the adjunct culture showed more rapid and more intense nutty flavor development than control cheeses. Cheeses that had higher intensities of nutty flavors also had a higher concentration of 2/3-methyl butanal and 2-methyl propanal compared with control cheeses, which again confirmed that these compounds are a source of nutty flavor in Cheddar cheese. Results from this study provide a simple methodology for cheese manufacturers to obtain consistent nutty flavor in Cheddar cheese.     

Effect of alpha-acetolactate decarboxylase inactivation on alpha-acetolactate and diacetyl production by Lactococcus lactis subsp. lactis biovar diacetylactis

Strains of Lactococcus lactis subsp. lactis biovar diacetylactis deficient in alpha-acetolactate decarboxylase produce alpha-acetolactate. This unstable compound is a precursor of acetoin and an aromatic compound, diacetyl. Following random mutagenesis of strain CNRZ 483, alpha-acetolactate decarboxylase-negative mutant 483 M1 was selected. When grown in milk, its growth and acidification characteristics were similar to those of the parental strain. In anaerobic conditions, the parental strain produced 2.10 mM acetoin and less than 0.05 mM diacetyl. The mutant accumulated up to 2.11 mM alpha-acetolactate, which spontaneously degraded to acetoin and diacetyl. After 24 h of culture, the alpha-acetolactate concentration was only 0.49 mM and the acetoin and diacetyl concentrations reached 1.50 mM and 0.26 mM, respectively. Diacetyl production by both strains increased in aerobic conditions, as well as when citrate was added. In contrast to cultures of the parental strain, however, diacetyl and acetoin concentrations in mutant cultures continued to increase without reaching a plateau. The results also showed that diacetyl production by wild type L. lactis subsp. lactis biovar diacetylactis strains cannot be explained uniquely by the spontaneous decarboxylation of the alpha-acetolactate produced in the culture medium.     

Technological characterization of bacteriocin producing Lactococcus lactis strains employed to control Listeria monocytogenes in cottage cheese

In recent years, there has been a particular focus on the application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives to control the growth of spoilage and pathogenic bacteria in food. Bacteriocins are antimicrobial peptides which can be added to foods in concentrated forms as food preservatives, e.g. additives, or they can be produced in situ by starters or protective cultures. In this study, twenty Lactococcus lactis bacteriocin producers previously isolated from Italian fermented foods were subjected to a variety of physical and biochemical tests in order to identify those with the greatest potential as starter cultures in cheese production. Of these, four strains isolated from cheese (one nisin Z producer, one nisin A producer and two lacticin 481 producers) which fulfilled the desired technological criteria were assessed for their ability to control Listeria monocytogenes. The subsequent application of these bacteriocinogenic strains as starter cultures in Cottage cheese established that the nisin A producing Lact. lactis 40FEL3, and to a lesser extent the lacticin 481 producers 32FL1 and 32FL3, successfully controlled the growth of the pathogen. This is the first study to directly compare the ability of nisin A, nisin Z and lacticin 481 producing strains to control listerial growth during the manufacture and storage of Cottage cheese.     

Inhibition of a methicillin-resistant Staphylococcus aureus strain in Afuega'l Pitu cheese by the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729

Methicillin-resistant Staphylococcus aureus strains are a potential threat for food safety because foodborne illness caused by methicillin-resistant Staphylococcus aureus has been reported even though these strains were only associated with nosocomial infections until recently. This article focuses on the inhibitory effect of the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729 on the growth of Staphylococcus aureus CECT 4013, a methicillin-resistant strain. S. aureus was inhibited by the presence of the nisin producer IPLA 729 in buffered Trypticase soy broth, milk, and Afuega'l Pitu cheese, an acid-coagulated cheese manufactured in Asturias, Northern Spain. A reduction of 3.66 log units was observed in Trypticase soy broth at the end of the incubation period. In milk, viable counts of S. aureus were undetectable or were reduced by 2.16 log units in 24 h depending on the initial inoculum (1.8 x 10(4) and 7.2 x 10(6) CFU/ml). The staphylococcal strain was also undetected in test cheeses in which the nisin Z producer was present whereas 2 log units were detected in control cheeses at the end of ripening.     

Control of late blowing in cheese by adding lacticin 3147-producing Lactococcus lactis IFPL 3593 to the starter

Bacteriocins produced by lactic acid bacteria offer potential as tools for ensuring food safety and quality. Lacticin 3147 is a two-peptide lantibiotic previously shown to inhibit a broad range of food spoilage and pathogenic bacteria. Lacticin 3147-producing Lactococcus lactis IFPL 3593 was investigated for its ability to inhibit the growth of clostridia as a means to prevent late blowing in cheese. L. lactis IFPL 3593 was shown to inhibit germination of clostridia spores and prevent late blowing in semi-hard cheeses, with a 5 log g^?1 reduction in the numbers of spores when compared to control cheeses. Furthermore, this bacteriocin-producing strain demonstrated considerable potential as a biopreservative agent against heterofermentative lactobacilli and their associated blowing defects. The use of this strain to prevent late blowing in cheese thereby represents a promising alternative to the addition of lysozyme particularly given the increasing concerns regarding the potential allergenicity of this additive.     

Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama

Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.     

成熟温度对牦牛乳硬质干酪中乳酸菌自溶及氨肽酶活性的影响

通过动态监测5、10、15℃成熟牦牛乳硬质干酪在1~6个月成熟过程中乳酸菌总数、乳酸脱氢酶和氨肽酶活性等变化,探究提高成熟温度对其乳酸菌自溶和氨肽酶活性的影响。研究结果表明:成熟温度对p H值影响显著(P0.05)。干酪中乳酸菌在1~6个月成熟过程中其数量呈现降低趋势。p H值与乳酸菌总数之间存在负相关性。5、10、15℃成熟干酪中乳酸脱氢酶活力依次呈现增加趋势,15、10℃成熟干酪中氨肽酶活性高于5℃成熟干酪中氨肽酶活性。因此,15℃成熟干酪中乳酸菌自溶程度最强,其次为10℃成熟干酪,5℃成熟干酪中乳酸菌自溶程度最弱。15℃成熟干酪中广谱性氨肽酶(broad-specificity aminopeptidase,Pep N)和脯氨酸氨肽酶(X-prolyl-dipeptidlylaminopeptidase,Pep X)活性分别是10℃成熟干酪中Pep N和Pep X活性的2.19和2.64倍。5℃成熟干酪中Pep N、Pep X活性较低,分别没有超过0.29 U/g和0.4 U/g。