Nonenzymatic Browning of Mozzarella Cheese

Mozzarella cheese was made with combinations of Streptococcus thermophilus nongalactose (Gal^?) and galactose fermenting (Gal⁺) strains and Lactobacillus helveticus (Gal⁺) and L. bulgaricus (Gal^?). Galactose was found in all Mozzarella cheese regardless of the culture used. The highest concentration was in cheese made with Streptococcus thermophilus Gal^?L. bulgaricus Gal^? combination with Streptococcus thermophilus Gal⁺Lactobacillus helveticus Gal⁺ having the least. Little if any lactose was found in any of the cheeses. The temperature and time during stretching of the curd inhibited fermentation of the residual galactose.Galactose accumulated in Mozzarella when either strain of Streptococcus thermophilus was used. The fermentation of accumulated galactose was the result of metabolism by Lactobacillus helveticus. There was a positive correlation between galactose content and brown color intensity when Mozzarella cheese was heated. A predictive test was effective in evaluating the browning tendency of cheese.     

Enhancement of flavour development in ras cheese made by direct acidification

An attempt has been made to enhance flavour development in Ras cheese made from directly acidified milk. Addition of a ripened cheese slurry, yoghurt culture (Streptococcus thermophilus + Lactobacillus bulgaricus) or cheese starter (S. lactis + L. casei + Leuconostoc citrovorum) to the chemically acidified curd enhanced flavour intensity, body characteristics, the formation of both soluble nitrogen compounds and Free Fatty Acids and stimulated bacterial growth. Sensory properties (or characteristics) of cheese from chemically acidified curd incorporating the above additives approached those of control cheese.     

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.     

Influence of a bacteriocin-producing lactic culture on proteolysis and texture of Hispánico cheese

Hispánico cheese was manufactured in duplicate experiments, each consisting of two 50-L vats, and ripened for 75 days. Lactic cultures for experimental cheese were 0.5% Lactococcus lactis subsp. lactis INIA 415 (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain), a bacteriocin-producing (Bac⁺) strain harbouring the structural genes of nisin Z and lacticin 481, 0.5% L. lactis subsp. lactis INIA 415-2, a Bac⁻ mutant and 2% TA052, a commercial Streptococcus thermophilus culture. Lactic cultures for control cheese were 1% L. lactis subsp. lactis INIA 415-2 and 2% TA052. S. thermophilus counts were lower, and levels of cell-free intracellular aminopeptidases higher, from day 1 in cheese made with the bacteriocin producer, indicating early lysis of the thermophilic culture. Experimental cheese showed reduced proteolysis of α_s-casein and lower levels of hydrophilic and hydrophobic peptides than control cheese. However, proteolysis as estimated by the o-phthaldialdehyde method and total level of free amino acids were in experimental cheese 1.38- and 2.47-fold, respectively, those in control cheese on day 25, and 1.49- and 2.34-fold, respectively, on day 75. Higher values of fracturability, elasticity and hardness were recorded from day 50 for cheese made with the bacteriocin producer, which were related to its higher residual α_s-casein content. The use of a bacteriocin-producing culture, though retarding α_s-casein proteolysis and softening of texture, enhanced considerably secondary proteolysis during cheese ripening.     

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.     

Effect of coagulant type and level on the properties of half-salt,half-fat Cheddar cheese made with or without adjunct starter: Improving texture and functionality

The potential of increasing proteolysis as a means of enhancing the texture and heat-induced flow of half-fat, half-salt Cheddar cheese made with control culture (CL, Lactococcus lactis subsp. cremoris/lactis) or adjunct culture (AC, CL + Lactobacillus helveticus) was investigated. Proteolysis was altered by substituting bovine chymosin (BC) with camel chymosin (CC), or by a 2.5-fold increase in level of BC. In cheese with CL-culture, increasing BC led to a large increase in pH and more rapid degradation of α_S1-casein during maturation, and cheese that was less firm after 180 d. In contrast, substitution of BC with CC in cheeses made with CL-culture had an opposite effect. While chymosin type and level had a similar influence on α_S1-casein hydrolysis in the AC-culture cheeses, it did not affect texture or flowability. Grading indicated that cheese made with AC-culture and with a higher level of BC was the most appealing.     

Use of Galactose-Fermenting Streptococcus thermophilus in the Manufacture of Swiss,Mozzarella, and Short-Method Cheddar Cheese

Galactose-fermenting (galactose-positive) strains of Streptococcus thermophilus, alone and combined with galactose-positive and galactose-negative strains of Lactobacillus bulgaricus, were used as starter cultures in the manufacture of Swiss and Mozzarella cheese and were paired with Streptococcus lactis (also galactose-positive) in short-method Cheddar cheese manufacture. Experimental Swiss cheese made with the galactose-positive Streptococcus thermophilus starter alone contained a large amount of galactose (ca. 26 to 28 µmol/g of curd) 28 h after hooping compared with control Swiss (< 2 µmol/g) made with a nongalactose fermenting strain of Streptococcus thermophilus and a galactose-positive strain of Lactobacillus bulgaricus. Mozzarella and short-method Cheddar made with only galactose-positive Streptococcus thermophilus also contained large amounts of galactose. Swiss cheese made with a galactose-positive strain of Streptococcus thermophilus and a galactose-negative strain of Lactobacillus bulgaricus had little galactose remaining after 28 h, indicating that the Lactobacillus had a stimulatory effect on galactose metabolism in Streptococcus thermophilus. These results indicate that galactose-fermenting Streptococcus thermophilus may have limited potential when used as single strain starter cultures in Swiss cheese, but may be useful when combined with galactose-positive Lactobacillus in the manufacture of Mozzarella cheese.     

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.     

Application of exopolysaccharide-producing cultures in reduced-fat Cheddar cheese: composition and proteolysis

Proteolysis during ripening of reduced fat Cheddar cheeses made with different exopolysaccharide (EPS)-producing and nonproducing cultures was studied. A ropy strain of Lactococcus lactis ssp. cremoris (JFR1) and capsule-forming nonropy and moderately ropy strains of Streptococcus thermophilus were used in making reduced-fat Cheddar cheese. Commercial Cheddar starter was used in making full-fat cheese. Results showed that the actual yield of cheese made with JFR1 was higher than that of all other reduced-fat cheeses. Cheese made with JFR1 contained higher moisture, moisture in the nonfat substance, and residual coagulant activity than all other reduced-fat cheeses. Proteolysis, as determined by PAGE and the level of water-soluble nitrogen, was also higher in cheese made with JFR1 than in all other cheeses. The HPLC analysis showed a significant increase in hydrophobic peptides (causing bitterness) during storage of cheese made with JFR1. Cheese made with the capsule-forming nonropy adjunct of S. thermophilus, which contained lower moisture and moisture in the nonfat substance levels and lower chymosin activity than did cheese made with JFR1, accumulated less hydrophobic peptides. In conclusion, some EPS-producing cultures produced reduced-fat Cheddar cheese with moisture in the nonfat substance similar to that in its full-fat counterpart without the need for modifying the standard cheese-making protocol. Such cultures might accumulate hydrophobic (bitter) peptides if they do not contain the system able to hydrolyze them. For making high quality reduced-fat Cheddar cheese, EPS-producing cultures should be used in conjunction with debittering strains.     

The effect of sodium reduction with and without potassium chloride on the survival of Listeria monocytogenes in Cheddar cheese

Sodium chloride (NaCl) in cheese contributes to flavor and texture directly and by its effect on microbial and enzymatic activity. The salt-to-moisture ratio (S/M) is used to gauge if conditions for producing good-quality cheese have been met. Reductions in salt that deviate from the ideal S/M range could result in changing culture acidification profiles during cheese making. Lactococcus lactis ssp. lactis or Lc. lactis ssp. cremoris are both used as cultures in Cheddar cheese manufacture, but Lc. lactis ssp. lactis has a higher salt and pH tolerance than Lc. lactis ssp. cremoris. Both salt and pH are used to control growth and survival of Listeria monocytogenes and salts such as KCl are commonly used to replace the effects of NaCl in food when NaCl is reduced. The objectives of this project were to determine the effects of sodium reduction, KCl use, and the subspecies of Lc. lactis used on L. monocytogenes survival in stirred-curd Cheddar cheese. Cheese was manufactured with either Lc. lactis ssp. lactis or Lc. lactis ssp. cremoris. At the salting step, curd was divided and salted with a concentration targeted to produce a final cheese with 600 mg of sodium/100 g (control), 25% reduced sodium (450 mg of sodium/100 g; both with and without KCl), and low sodium (53% sodium reduction or 280 mg of sodium/100 g; both with and without KCl). Potassium chloride was added on a molar equivalent to the NaCl it replaced to maintain an equivalent S/M. Cheese was inoculated with a 5-strain cocktail of L. monocytogenes at different times during aging to simulate postprocessing contamination, and counts were monitored over 27 or 50 d, depending on incubation temperature (12 or 5°C, respectively). In cheese inoculated with 4 log₁₀ cfu of L. monocytogenes/g 2 wk after manufacture, viable counts declined by more than 3 log₁₀ cfu/g in all treatments over 60 d. When inoculated with 5 log₁₀ cfu/g at 3 mo of cheese age, L. monocytogenes counts in Cheddar cheese were also reduced during storage, but by less than 1.5 log₁₀ cfu/g after 50 d. However, cheese with a 50% reduction in sodium without KCl had higher counts than full-sodium cheese at the end of 50 d of incubation at 4°C when inoculated at 3 mo. When inoculated at 8 mo postmanufacture, this trend was only observed in 50% reduced sodium with KCl, for cheese manufactured with both cultures. This enhanced survival for 50% reduced-sodium cheese was not seen when a higher incubation temperature (12°C) was used when cheese was inoculated at 3 mo of age and monitored for 27 d (no difference in treatments was observed at this incubation temperature). In the event of postprocessing contamination during later stages of ripening, L. monocytogenes was capable of survival in Cheddar cheese regardless of which culture was used, whether or not sodium had been reduced by as much as 50% from standard concentrations, or if KCl had been added to maintain the effective S/M of full-sodium Cheddar cheese.     

Flavour enhancement of low-fat Feta-type cheese using a commercial adjunct culture

The effect of a commercial adjunct culture (CR-213), containing Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp.lactis, added at the level of 0.06 or 0.09% (w/w) to cheese milk, on the characteristics of the resultant low-fat Feta-type cheese during aging, was studied. Two controls, a full-fat cheese (∼22% fat) and a low-fat cheese (∼7% fat, made using the standard procedure), were also prepared. The results indicated that the adjunct containing low-fat cheeses exhibited no significant (P>0.05) differences in compositional (moisture, fat, protein, salt, pH) or textural (force and compression to fracture, hardness) characteristics in comparison with the low-fat control cheese. It was also found that the use of the adjunct culture slightly improved the flavour intensity of the low-fat cheese which received a flavour score similar to that of the full-fat control cheese. Moreover, the experimental low-fat cheeses received significantly (P<0.05) higher total scores (overall quality) than the low-fat control cheese but lower than the full-fat cheese.     

Antimicrobial activity of pediocin-producing Lactococcus lactis on Listeria monocytogenes,Staphylococcus aureus and Escherichia coli O157:H7 in cheese

The antimicrobial activity of two pediocin-producing transformants obtained from wild strains of Lactococcus lactis on the survival of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 during cheese ripening was investigated. Cheeses were manufactured from milk inoculated with the three pathogens, each at approximately 6 log cfu mL⁻¹. Pediococcus acidilactici 347 (Ped⁺), Lc. lactis ESI 153, Lc. lactis ESI 515 (Nis⁺) and their respective pediocin-producing transformants Lc. lactis CL1 (Ped⁺) and Lc. lactis CL2 (Nis⁺, Ped⁺) were added at 1% as adjuncts to the starter culture. After 30 d, L. monocytogenes, S. aureus and E. coli O157:H7 counts were 5.30, 5.16 and 4.14 log cfu g⁻¹ in control cheese made without adjunct culture. On day 30, pediocin-producing derivatives Lc. lactis CL1 and Lc. lactis CL2 lowered L. monocytogenes counts by 2.97 and 1.64 log units, S. aureus by 0.98 and 0.40 log units, and E. coli O157:H7 by 0.84 and 1.69 log units with respect to control cheese. All cheeses made with nisin-producing LAB exhibited bacteriocin activity throughout ripening. Pediocin activity was only detected throughout the whole ripening period in cheese with Lc. lactis CL1. Because of the antimicrobial activity of pediocin PA-1, its production in situ by strains of LAB growing efficiently in milk would extend the application of this bacteriocin in cheese manufacture.     

Isolation and characterisation of exopolysaccharide-producing Weissella and Lactobacillus and their application as adjunct cultures in Cheddar cheese

This study characterised exopolysaccharide-producing lactic acid bacteria and examined their potential for use in Cheddar cheese manufacture. Two strains were chosen for incorporation as adjunct cultures in Cheddar cheese manufacture: namely, the homopolysaccharide-producers Weissella cibaria MG1 and Lactobacillus reuteri cc2. These strains both produce dextrans with molecular masses ranging from 10⁵ to 10⁷ Da. Both strains were used in the production of miniature Cheddar cheeses that employed a conventional commercial cheese starter culture Lactococcus lactis R604. A cheese was also included that used purified dextran as an ingredient. The W. cibaria strain survived in cheese with levels increasing by 1.5 log cycles over the ripening period. All experimental cheeses (adjunct or exopolysaccharide ingredient) had higher moisture levels compared with the control cheese made using starter alone. Inclusion of the adjunct strains had no detectable negative effects on cheeses in terms of proteolysis.     

Flavour precursor development in Cheddar cheese due to lactococcal starters and the presence and lysis of Lactobacillus helveticus.

The rapid release of intracellular enzymes due to autolysis of lactic acid bacteria in the cheese matrix has been shown to accelerate cheese ripening. The objective of this work was to investigate the evolution of the flavour precursors, individual free amino acids (FAAs), free fatty acids (FFAs) and volatile compounds that contribute to the sensory profiles of cheeses at 2, 6 and 8 months of ripening in Cheddar cheese manufactured using starter systems which varied with respect to their autolytic properties. Starter system A contained a blend of two commercial Lactococcus lactis strains (223 and 227) which had a low level of autolysis. System B was identical to A but also included a highly autolytic strain of Lactobacillus helveticus (DPC4571). System C contained only strain DPC4571. Levels of all individual FAAs were elevated in cheeses B and C relative to A after 2 months of ripening. By 8 months of ripening the main FAA were glutamate, leucine, lysine, serine, proline and valine. Levels of C_6:0, C_8:0, C_12:0 and C_18:0 fatty acids did not vary greatly over ripening, while levels of C_4:0, C_10:0, C_14:0, C_16:0 and C_18:1 were elevated in cheeses B and C. Principal component analysis of the headspace volatiles separated cheese A from cheeses B and C. Cheeses B and C had highest levels of dimethyl disulphide, carbon sulphide, heptanal, dimethyl sulphide, ethyl butanoate, 2-butanone, and 2-methyl butanal and were described as having a ‘caramel’ odour and ‘sweet’, ‘acidic’ and ‘musty’ flavour. Cheese A had highest levels of 2-butanol, 2-pentanone, 2-heptanone, 1-hexanol and heptanal and was described as having a ‘sweaty/ sour’ odour and ‘soapy’, ‘bitter’ and ‘mouldy’ flavour. The results highlight the impact of starter lactococci on flavour precursor development and the positive effect of Lb. helveticus and the lysis of this strain on enhancing levels of substrate and flavour precursors early during ripening resulting in early flavour development.     

Production of volatile aroma compounds by kefir starter cultures

Production of carbonyl compounds by single-strain cultures, kefir starter (Lactobacillus delbrueckii subsp. bulgaricus HP1+Lb. helveticus MP12+Lactococcus lactis subsp. lactis C15+Streptococcus thermophilus T15+Saccharomyces cerevisiae A13) and kefir grains during fermentation and storage of kefir was studied. The content of carbonyl compounds produced by kefir starter was greater than that produced by kefir grains. The maximum acetaldehyde concentration (18.3 μg g⁻¹) in kefir with starter culture was mainly due to the metabolic activity of Lb. delbrueckii subsp. bulgaricus HP1 isolated from kefir grains. The highest diacetyl production activity was recorded in the starter culture (1.87 μg g⁻¹) and the single-strain culture St. thermophilus T15 (1.62 μg g⁻¹), followed by Lb. helveticus MP12 (0.85 μg g⁻¹) and Lc. lactis subsp. lactis C15 (0.42 μg g⁻¹). The lactobacilli Lb. delbrueckii subsp. bulgaricus HP1 and Lb. helveticus MP12 produced acetone, which was not found in the cocci cultures. The presence of 2-butanone was related to the production ability of Lb. helveticus MP12. In comparison, Lc. lactis subsp. lactis C15 synthesized ethyl acetate more actively than the other single-strain cultures included in the starter. S. cerevisiae A13 produced ethanol and CO₂ in amounts (3975 μg g⁻¹; 1.80 g L⁻¹) that lent cultured kefir distinctive flavour and aroma characteristic of authentic kefir.     

Assessment of expression of Leloir pathway genes in wild-type galactose-fermenting Streptococcus thermophilus by real-time PCR

In this study, four galactose-positive (Gal⁺) Streptococcus thermophilus strains viz. AJM, JM1, KM3 and AUKD8 and one galactose-negative (Gal^?) S. thermophilus NCDC 218 were used to characterize the organization of Leloir pathway genes using long-range PCR, and expression of these genes were studied using real-time PCR, in presence of different sugars. Long-range PCR results showed that both Gal⁺ and Gal^? isolates, the gal–lac gene order (galRKTEM–lacSZ), are conserved including the size of individual genes. The promoter sequence of the three Gal⁺ isolates (AJM, JM1 and KM3) possessed single base pair deletion at ?28 region of galR and C to T substitution at ?9 box galK region. In contrast, Gal⁺ AUKD8 had A to T substitution at preceding ?25 region of galR. The expression of galK and galM grown in the presence of galactose was significantly higher in case of AJM (30- and 7.6-fold, respectively), followed by KM3 and JM1. In addition, galR, galT and galE showed higher expression in galactose, than in lactose and glucose medium. This study gives a preliminary idea on Leloir pathway gene expression in wild Gal⁺ S. thermophilus, and further studies may throw more light on the role of gal–lac operon in galactose metabolism.     

The conjugal plasmid pLL10236 encodes lactose fermentation ability,restriction/modification activity,bacteriocin production and immunity in Lactococcus lactis subsp. lactis LL102

A 36 kb plasmid, designed as pLL 10236, was determined in Lactococcus lactis subsp.Lactis LL102. This plasmid mediates lactose fermentation ability (Lac⁺), bacteriocin production (Bac⁺) and immunity (Imm⁺), and restriction/modification (R⁺M⁺) activity against bacteriophage Ø1120 in strain LL102. The conjugal ability of pLL 10236 and expression of pLL 10236 encoding traits were determined by using a Lac⁻, Bac⁻, Imm⁻and (R⁻M⁻) recipient strain L. lactis subsp. lactis P81-1.