Quantitative measurement of tetrahydromenaquinone-9 in cheese fermented by propionibacteria

Propionibacteria produce tetrahydromenaquinone-9 [MK-9 (4H)] as a major menaquinone (vitamin K₂). This study aimed to determine the MK-9 (4H) concentration in commercial propionibacteria-fermented cheese. The MK-9 (4H) concentration was quantified using an HPLC instrument with a fluorescence detector after postcolumn reduction. Among the various cheese samples, the MK-9 (4H) concentration was highest in Norwegian Jarlsberg cheese, followed by Swiss Emmental cheese. In contrast, the MK-9 (4H) concentrations in Appenzeller or Gruyère cheeses were extremely low or undetected. Likewise, the concentrations in Comte and Raclette cheeses were lower than those in Jarlsberg and Emmental cheeses. In the present study, the MK- 9 (4H) concentration in cheese showed a correlation with the viable propionibacterial cell count and propionate concentration. This implies that the increase in propionibacteria contributed to the generation of MK-9 (4H) in cheese. We presumed, based on these results, that Swiss Emmental and Norwegian Jarlsberg cheeses contain a meaningful amount of vitamin K because of their high MK-9 (4H) concentrations (200 to 650 ng/g).     

Subspecies-Specific Nested PCR Assay for Detection of Lactococcus lactis spp. lactis and spp. cremoris

A nested PCR-based assay composed of Lactococcus lactis species-specific primers for the nest 1 amplification and subspecies-specific primers for the nest 2 amplification was validated with the identified strains of L. lactis isolated from dairy and nondairy sources and positive and negative control strains. Forward and reverse primer set was designed for nest 1 amplification targeting the conserved housekeeping gene yueF encoding nonproteolytic protein from peptidase family M16 of L. lactis. Amplicons of 447 bp of yueF were subjected for nest 2 amplification producing amplicons of 372 bp. The designed outer primer set for nest 1 amplification was observed to be specific to L. lactis because the DNA from other bacteria could not be amplified and the inner primer set for nest 2 amplification was found to be specific for the detection of ssp. lactis and cremoris of L. lactis.     

Impact of ropy and capsular exopolysaccharide-producing strains of Lactococcus lactis subsp. cremoris on reduced-fat Cheddar cheese production and whey composition

Cheddar cheese mixed starter cultures containing exopolysaccharide (EPS)-producing strains of Lactococcus lactis subsp. cremoris (Lac. cremoris) were characterized and used for the production of reduced-fat Cheddar cheese (15% fat). The effects of ropy and capsular strains and their combination on cheese production and physical characteristics as well as composition of the resultant whey samples were investigated and compared with the impact of adding 0.2% (w/v) of lecithin, as a thickening agent, to cheese milk. Control cheese was made using EPS-non-producing Lac. cremoris. Cheeses made with capsular or ropy strains or their combination retained 3.6–4.8% more moisture and resulted in 0.29–1.19 kg/100 kg higher yield than control cheese. Lecithin also increased the moisture retention and cheese yield by 1.4% and 0.37%, respectively, over the control cheese. Lecithin addition also substantially increased viscosity, total solid content and concentrating time by ultra-filtration (UF) of the whey produced. Compared with lecithin addition, the application of EPS-producing strains increased the viscosity of the resultant whey slightly, while decreasing whey total solids, and prolonging the time required to concentrate whey samples by UF. The amount of EPS expelled in whey ranged from 31 to 53 mg L⁻¹. Retention of EPS-producing strains in cheese curd was remarkably higher than that of non-producing strains. These results indicate the capacity of EPS-producing Lac. cremoris for enhanced moisture retention in reduced-fat Cheddar cheese; these strains would be a promising alternative to commercial stabilizers.     

The effect of NaCl and metabolic profile of propionibacteria on eye formation in experimental Swiss-type cheese

The influence of three brining times (0, 1, and 3 d) and two propionibacteria (PAB) cultures (Prop A and Prop B) on eye formation was investigated in experimental Swiss-type cheeses by comparing PAB counts, biochemical parameters, eye numbers, diameters and volumes. Prop A was strongly inhibited with increasing NaCl content. As a result, eye volume decreased considerably, as well as eye diameter especially towards the cheese surface. Prop B exclusively converted l-lactate and was much less affected by increasing NaCl content, hence, eye formation changed less. The salt sensitivity of the cultures also influenced the spatial distribution of the eyes. The width of the eyeless border zone was mainly affected by three factors: CO₂ diffusion to the outside, propionic acid fermentation, and body firmness. The study clearly showed that strain-specific salt sensitivity of PAB is an important feature that strongly influences eye formation in Swiss-type cheeses with different salt content.     

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.     

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.     

Nisin-producing Lactococcus spp. from mayonnaise-based products and their raw materials

Three bacteriocins were purified from lactic acid bacteria isolated from mayonnaise-based finished products and the raw materials used in the production of the finished products. All three bacteriocin-producing strains (EN3a, EN14a, and EN15b) were identified by sequencing of the 16S rDNA as Lactococcus lactis. The RP-HPLC fractions showed strong antimicrobial activity against Listeria monocytogenes. Mass spectrometry analysis of active fractions showed that all strains produced nisin A (3,353.7 Da). These results were confirmed by amplification of nisin structural gene by PCR, by lack of activity against a nisin-producing strain, and by total loss of activity after reaction of purified bacteriocins with glutathione. The strains Lactococcus lactis EN3a, EN14a, and EN15b are the first nisin-producing strains isolated from mayonnaise-based commercial products and from the raw materials used in their production. The ability of these strains to produce nisin could be useful to improve food quality of these and related products.     

Assessment of potential probiotic properties of Lactobacillus spp., Lactococcus spp., and Pediococcus spp. strains isolated from kefir

In this study, the metabolic activities (in terms of quantities of the produced lactic acid, hydrogen peroxide, and exopolysaccharides) of 8 strains of Lactobacillus spp., Lactococcus spp., and Pediococcus spp., were determined. Lactic acid levels produced by strains were 8.1 to 17.4 mg/L. The L. acidophilus Z1L strain produced the maximum amount (3.18 μg/mL) of hydrogen peroxide. The exopolysaccharides (EPS) production by the strains was ranged between 173 and 378 mg/L. The susceptibility of 7 different antibiotics against these strains was also tested. All strains were found to be sensitive to ampicillin. The tolerance of the strains to low pH, their resistance to bile salts of strains, and their abilities to autoaggregate and coaggregate with Escherichia coli ATCC 11229 were also evaluated. High EPS-producing strains showed significant autoaggregation and coaggregation ability with test bacteria (P < 0.01). A correlation also was determined between EPS production and acid-bile tolerance (P < 0.05). EPS production possibly affects or is involved in acid-bile tolerance and aggregation of Lactobacillus spp., Lactococcus spp., and Pediococcus spp. strains and supports the potential of L. acidophilus Z1L strain as new probiotic.     

Growth inhibition of Listeria spp. on Camembert cheese by bacteria producing inhibitory substances.

Bacterial strains exhibiting antimicrobial activity towards other bacteria are quite common in nature. During the past few years several genera have been shown to exert inhibitory action against Listeria. spp. In the present work strains of Enterococcus, Lactobacillus and Lactococcus were tested for their influence on the development of Listeria spp. on Camembert cheese. Partial or complete inhibition of growth of Listeria spp. was observed using various inhibitory bacteria. Complete inhibition occurred when the inhibitory strain was used as a starter culture and there was a low level of contamination with Listeria spp. during the first stage of ripening. Very little inhibition occurred if the inhibitory strain was added together with the starter culture.     

Accelerating cheese proteolysis by enriching Lactococcus lactis proteolytic system with lactobacilli peptidases

The knowledge available on the genetics and proteolytic system of lactic acid bacteria makes it possible to genetically engineer starters with increased proteolytic properties. Our objective was to identify the best available strains capable of accelerating or modulating casein proteolysis during cheese ripening.To attain this goal, we used Lactococcus lactis strains expressing 5 different Lactobacillus peptidases to ripen a cheese model. At the end of ripening, free amino acids were quantitatively and qualitatively analysed.We identified the mixture of prolidase, PepQ, and X-prolyl dipeptidyl peptidase, PepX, as well as the peptidase PepW as the most efficient peptidases to increase, up to 3-fold, the overall level of amino acids at the end of ripening. The levels of threonine, asparagine, glycine, methionine, valine, glutamine, isoleucine and proline in particular increased (more than 3.5 fold). Grouping the amino acids produced according to the specific aroma compounds that each may give rise to following an enzymatic or chemical conversion, revealed that expression of PepW or PepX and PepQ increased the amounts of all groups of amino acids while expression of PepQ or PepN increased more especially those of aromatic amino acids/proline and glutamic acid, respectively.The combination of increased proteolysis and conversion of amino acids into aroma compounds now needs to be tested. In addition, the role of proline and its derived compounds in the overall flavour of cheese should be investigated.     

Effect of feeding propionibacteria on milk production by early lactation dairy cows

This experiment was conducted to determine the effect of a direct-fed microbial agent, Propionibacterium strain P169 (P169), on rumen fermentation, milk production, and health of periparturient and early-lactation dairy cows. Starting 2 wk before anticipated calving, cows were divided into 2 groups and fed a control diet or the control diet plus 6 × 10¹¹ cfu/d of P169. Cows were changed to a lactation diet at calving, and treatments continued until 119 d in milk. Rumen fluid samples were taken about 1 wk before calving, and at 1 and 14 wk after calving. Cows fed P169 had lower concentrations of acetate (mol/100 mol of total volatile fatty acids) at all time points, greater concentrations of propionate on the first and last sampling points, and greater concentrations of butyrate on the first 2 time points. Concentrations of glucose in plasma and milk and plasma concentrations of β-hydroxybutyrate were not affected by treatment. Cows fed P169 had greater concentrations of plasma nonesterified fatty acids on d 7 of lactation. The high nonesterified fatty acids at that time point was probably related to the high production of milk during that period by cows fed the additive. Cows fed P169 during the first 17 wk of lactation produced similar amounts of milk (44.9 vs. 45.3 kg/d, treatment vs. control) with similar composition as cows fed the control diet. Calculated net energy use for milk production, maintenance, and body weight change was similar between treatments, but cows fed the P169 consumed less dry matter (22.5 vs. 23.5 kg/d), which resulted in a 4.4% increase in energetic efficiency.     

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.     

Occurrence of nisin Z production in Lactococcus lactis BFE 1500 isolated from wara, a traditional Nigerian cheese product

Screening for bacteriocin production of 500 strains of lactic acid bacteria (LAB) from various African fermented foods resulted in the detection of a bacteriocin producing Lactococcus lactis (BFE 1500) isolated from a dairy product called wara. The bacteriocin inhibited not only the closely related LAB, but also strains of Listeria monocytogenes, Listeria innocua, Clostridium butyricum, Clostridium perfringens, Bacillis cereus and Staphylococcus aureus. It was heat stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10), but highest activity was observed in the lower pH range. The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K, but not by other proteases. Growth kinetic assay indicated stronger growth inhibition by the bacteriocin produced by Lc. lactis BFE 1500 on L. monocytogenes WS 2250 and B. cereus DSM 2301 than with the nisin A producing strain DSM 20729. Polymerase chain reaction indicated the presence of the nisin operon in strain BFE 1500 and sequencing of its structural gene showed that Lc. lactis BFE 1500 produced the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The genetic determinants for bacteriocin production in strain BFE 1500 are located on a conjugative transposon. The ability of the bacteriocin produced by Lc. lactis BFE 1500 to inhibit a wide range of food-borne pathogens is of special interest for food safety, especially in the African environment with perennial problems of poor food hygiene.     

Citrinin production and stability in cheese

Citrinin is a nephrotoxic fungal metabolite that has been demonstrated to be mutagenic in hepatocytes. It can be produced by several fungal species that belong mainly to the genus Penicillium and has been isolated from many feeds and human foods. Cheese is a very sensitive product because it can be naturally contaminated by citrinin-producing molds. The purpose of this study was to determine whether citrinin can be produced in cheeses and whether it is stable in these products. Both toxigenic strains of Penicillium citrinum and Penicillium expansum used were able to produce citrinin in cheese at 20 degrees C, but not at 4 degrees C. Up to 600 mg of citrinin per kg of cheese was obtained after 10 days of incubation. Interestingly, fresh goat cheese appeared to be a more favorable substrate for toxigenesis than did yeast extract-sucrose medium. Although contamination was mainly superficial, 33% of the toxin remained in cheese after trimming. Moreover, citrinin appeared to be very stable in some of the tested cheeses (goat cheese, Saint Marcellin, Soignon). For all cheeses tested, more than 50% of the initial content of citrinin was still present after 8 days of storage. Taken together, these results suggest that the contamination of cheeses by wild strains of Penicillium must be avoided.     

Stimulation of Cadaverine Production by Foodborne Pathogens in the Presence of Lactobacillus,Lactococcus, and Streptococcus spp.

Abstract: The effect of Lactobacillus plantarum (FI8595), Lactococcus lactis subsp. cremoris MG 1363), Lactococcus lactis subsp. lactis (IL 1403), and Streptococcus thermophilus on cadaverine and other biogenic amine production by foodborne pathogens was investigated lysine decarboxylase broth. Both of lactic acid bacteria and foodborne pathogens used (especially Staphylococcus aureus, E. coli, Lc. lactis subsp. lactis and Lb. plantarum) had an ability to convert aminoacids into biogenic amine. The conversion of lysine into cadaverine was the highest (167.11 mg/L) by Lactobacillus spp. Gram‐positive bacteria generally had a greater ability to produce cadaverine with corresponding value of 46.26, 53.76, and 154.54 mg/L for Enterococcus faecalis, S. aureus, and Listeria monocytogenes, respectively. Significant variations on biogenic amine production were observed in the presence of lactic acid bacteria strains (P < 0.05). The role of lactic acid bacteria on biogenic amine production by foodborne pathogens varied depending on strains and specific amine. Cadaverine accumulation by Enterobactericeae was increased in the presence of lactic acid bacteria strains except for St. thermophilus, which induced 2‐fold lower cadaverine production by S. Paratyphi A. Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris induced 10‐fold higher increases in histamine for E. coli and K. pneumoniae, respectively. Lactic acid bacteria resulted in strong increases in cadaverine production by P. aeruginosa, although remarkable decreases were observed for histamine, spermidine, dopamine, agmatine, and TMA in the presence of lactic acid bacteria in lysine decarboxylase broth . The result of the study showed that amine positive lactic acid bacteria strains in fermented food led to significant amine accumulation by contaminant bacteria and their accumulation in food product may be controlled by the use of proper starters with amine‐negative activity. Practical Application: Foodborne pathogens and certain lactic acid bacteria are particularly active in the production of biogenic amines. Most of the strains of bacteria possess more than 1 amino acid decarboxylase activity under lysine enrichment culture conditions. Lactic acid bacteria strains had a significant role on increase putrescine accumulation by foodborne pathogens. The increased production of biogenic amines in mixed culture is the result of presence of amine positive lactic acid bacteria strains. The addition of a proper selected starter culture with amine‐negative activity is advisable to produce safer fermented food with low contents of biogenic amines.     

Aroma compound production in cheese curd by coculturing with selected yeast and bacteria.

The microorganisms involved in cheese ripening produce various volatile compounds and induce typical flavors that contribute to cheese variety. To investigate aroma compound generation of cheese microflora, we used a dynamic headspace-gas chromatography-mass spectrometry analysis. To obtain good sensitivity and repeatability of quantification, dynamic headspace conditions and sample preparation were first optimized and led to an extraction set up in which samples were heated at 60 degrees C and diluted with water without pH adjustment. Then three different yeasts and three Geotrichum candidum commonly used in mold surface ripened cheeses were studied in pure culture in a cheese model medium. Thirty-nine cocultures of these three yeasts, the three G. candidum, and five bacteria were studied in the same medium to assess the interaction between microorganisms on aroma compound production. Twenty-four volatile compounds belonging to different chemical classes (alcohols, aldehydes, esters, sulfides, terpenes) were identified and quantified. Yeasts and especially Kluyveromyces lactis produced large amounts of alcohols, aldehydes, esters, and terpenes when cultured alone or in association. Geotrichum candidum and especially G. candidum strain G3 generated the largest amount of sulfides when cultured alone or in association. Finally, bacteria also produced aroma compounds but, except for Brevibacterium linens strain B5, which produced dimethyl trisulfide and ketones, no specific trend in the production of particular aroma compounds could be evidenced.     

Characterization of Clostridium spp. isolated from spoiled processed cheese products

Of 42 spoiled cheese spread products, 35 were found to harbor Clostridium spp. Typical signs of spoilage were gas production and off-odor. The identity was determined for about half of the isolates (n = 124) by Analytab Products (API), Biolog, the RiboPrinter System, 16S rDNA sequencing, cellular fatty acid analysis, or some combination of these. The majority of isolates were identified as Clostridium sporogenes (in 33% of products), but Clostridium cochlearium (in 12% of products) and Clostridium tyrobutyricum (in 2% of products) were also retrieved. Similarity analysis of the riboprint patterns for 21 isolates resulted in the identification of 10 ribogroups. A high degree of relatedness was observed between isolates of C. sporogenes originating from products produced 3 years apart, indicating a common and, over time, persistent source of infection. The spoilage potential of 11 well-characterized isolates and two culture collection strains was analyzed by inoculating shrimp cheese spread with single cultures and then storing them at 37 degrees C. Tubes inoculated with C. tyrobutyricum did not show any visible signs of growth (e.g., coagulation, discoloration, gas formation) in the cheese spread. After 2 weeks of incubation, tubes inoculated with C. cochlearium or C. sporogenes showed gas-holes, syneresis with separation of coagulated casein and liquid, and a change in color of the cheese. The amount of CO2 produced by C. cochlearium strains was approximately one-third that produced by the majority of C. sporogenes strains. To our knowledge, this is the first study to isolate and identify C. cochlearium as a spoilage organism in cheese spread.