Size-Exclusion HPLC Separation of Bitter and Astringent Fractions from Cheddar Cheese Made with Added Lactobacillus Strains to Accelerate Ripening

Lactobacillus strains were added as an adjunct to the regular lactic starter in Cheddar cheese manufacture in order to accelerate ripening. Microbial cheese proteolysis resulted in the release of free amino acids which were extracted with the astringent and bitter fractions and separated by size-exclusion and reversed-phase HPLC chromatography. Lactobacillus strains generally increased the degree of proteolysis. L. plantarum and L. brevis produced off-flavors possibly due to an accumulation of medium-size peptides. The control cheese (without lactobacilli) had the most peptides with a mean molecular- weight of < 1000 daltons and had a flavor described as slightly bitter. Addition of L. casei-casei L2A accelerated ripening and yielded a well-aged Cheddar cheese without any bitterness even after 7 months at 6°C.     

Impact of liposome-encapsulated enzyme cocktails on cheddar cheese ripening

Cheddar cheese proteolysis and lipolysis were accelerated using liposome-encapsulated enzymatic cocktails. Flavourzyme, neutral bacterial protease, acid fungal protease and lipase (Palatase M) were individually entrapped in liposomes and added to cheese milk prior to renneting. Flavourzyme was tested alone at three concentrations (Z1, Z2 and Z3 cheeses). Enzyme cocktails consisted of lipase and bacterial protease (BP cheeses), lipase and fungal protease (FP cheeses) or lipase and Flavourzyme (ZP cheeses). The resulting cheeses were chemically, rheologically and organoleptically evaluated during 3 months of ripening at 8 °C. Levels of free fatty acids and appearance of bitter and astringent peptides were measured. Certain enzyme treatments (BP and ZP) resulted in cheeses with more mature texture and higher flavor intensity in a shorter time compared with control cheeses. No bitter defect was detected except in 90-day-old FP cheese. A full aged Cheddar flavor was developed in Z3 and ZP cheeses, while treatment BP led to strong typical Cheddar flavor by the second month and did not exhibit any off-flavor when ripening was extended for a further month.     

Viscoelastic Properties of Cheddar Cheese: Effect of Calcium and Phosphorus,Residual Lactose,Salt-to-Moisture Ratio and Ripening Time

The viscoelastic properties of eight different types of Cheddar cheeses prepared with two levels of calcium (Ca) and Phosphorus (P) content, two levels of residual lactose content and two levels of salt to moisture ratio (S/M) ratio were studied in a STRESSTECH viscoanalyzer. The elastic (G′) and viscous (G″) modulus were measured at 0, 1, 2, 4, 6, and 8 months of ripening during heating the cheese samples from 30 to 70°C. Low levels of Ca and P content (0.53 g Ca and 0.39 g P /100 g cheese) in the Cheddar cheese resulted up to 20.9% and 15.9% lower elastic and viscous modulus respectively, compared to Cheddar cheese prepared with high levels of Ca and P content (0.67 g Ca and 0.53 g P/100g cheese) during ripening up to 8 months. Low levels of residual lactose (0.78 g/100g) in the Cheddar cheese resulted in 39.1 and 78.1% lower elastic and viscous modulus, respectively, compared to Cheddar cheese with high levels of residual lactose (1.4 g/100g) during ripening up to 8 months. In the same way, low levels of S/M ratio (4.8) in the Cheddar cheese resulted in 40.7 and 40.5% lower elastic and viscous modulus, respectively, compared to high levels of S/M ratio (6.4) during ripening up to 8 months. Upon heating from 30 to 70°C, the elastic and viscous modulus of the eight different types of Cheddar cheeses reduced up to 91.7 and 95.1%, respectively, during ripening. Cheddar cheese recorded maximum elastic modulus at the end of 8 months of ripening, and maximum viscous modulus at the end of 4 months of ripening.     

Dynamic Headspace Analyses of Volatile Compounds of Cheddar and Swiss Cheese during Ripening

The volatile compounds of Cheddar and Swiss cheeses during ripening for 9 wks at 11°and 21°C, respectively, were analyzed by a dynamic headspace analyzer/gas chromatograph every week. The compounds were identified by a combination of retention times and mass spectra. The volatile compounds of Cheddar increased 5.6 and Swiss cheese 15 times as ripening increased from 0 to 9 wks. The amount of volatile compounds of Swiss cheese was 2.6 times greater than that of Cheddar cheese during ripening. The volatile compounds were ketones, alcohols, aldehydes, esters, acids, sulfur compounds, benzenes, and hydrocarbons. Ketones and alcohols accounted for 92% of volatiles from Cheddar cheese and 88% of those from Swiss cheese.     

Influence of ripening temperature on the volatiles profile and flavour of Cheddar cheese made from raw or pasteurised milk

Cheddar cheeses were made from raw (R1, R8) or pasteurised (P1, P8) milk and ripened at 1°C (P1, R1) or 8°C (P8, R8). Volatile compounds were extracted from 6 month-old cheeses and analysed, identified and quantified by gas chromatography-mass-spectrometry. A detailed sensory analysis of the cheeses was performed after 4 and 6 months of ripening. The R8 cheeses had the highest and P1 the lowest concentrations of most of the volatile compounds quantified (fatty acids, ketones, aldehydes, esters, alcohols, lactones and methional). The R8 and P8 cheeses contained higher levels of most of the volatiles than R1 and P1 cheeses. Ripening temperature and type of milk influenced most of the flavour and aroma attributes. Principal component analysis (PCA) of aroma and flavour attributes showed that P1 and R1 had similar aroma and flavour profiles, while R8 had the highest aroma and flavour intensities, highest acid aroma and sour flavour. The age of cheeses influenced the perception of creamy/milky and pungent aromas. PCA of the texture attributes separated cheeses on the basis of ripening temperature. The R8 and P8 cheeses received significantly higher scores for perceived maturity than P1 and R1 cheeses. The P1 and R1 cheeses had similar values for perceived maturity. In a related study, it was found that concentrations of amino acids and fatty acids were similar in R1 and P1 during most of the ripening period, and R1 and P1 cheeses had low numbers of non-starter lactic acid bacteria (NSLAB). The panel found that ripening temperature, type of milk and age of cheeses did not influence the acceptability of cheese. It is concluded that NSLAB contribute to the formation of volatile compounds and affect the aroma and flavour profiles and the perceived maturity of Cheddar cheese.     

Effect of ripening temperature on the growth and significance of non-starter lactic acid bacteria in Cheddar cheese made from raw or pasteurised milk

Two cheese-making trials were conducted, each involving four cheeses, two made from raw milk (R1, R8) and two from pasteurised milk (P1, P8), and ripened at 1°C (R1, P1) or 8°C (R8, P8). The 1-day-old R1 and R8 cheese in trials 1 and 2 contained ∼10⁴ non-starter lactic acid bacteria (NSLAB) g⁻¹. In trial 1, no NSLAB were detected in 1-day-old P1 and P8 cheeses while those in trial 2 contained 10² cfu g⁻¹. In both trials, the maximum differences between the number of NSLAB in the cheeses ripened at 1 or 8°C were observed at 4 months, when the number of NSLAB in cheeses ripened at 8°C were 3 log cycles higher than in those ripened at 1°C. At the end of ripening (6-months), the number of NSLAB in P8 and R8 were ∼2 log cycles higher than in P1 and R1 cheeses, respectively. Primary proteolysis in the cheeses was markedly affected by ripening temperature, but not by pasteurisation of the cheese milk. Urea-polyacyrlamide gel electrophoretograms and reverse-phase (RP)-HPLC of the water-soluble fraction showed differences between cheeses made from raw or pasteurised milk and between cheeses ripened at 1 or 8°C. The concentration of amino acids and fatty acids were in the order R8>P8>R1>P1. Commercial graders awarded highest flavour scores to the R1 cheeses during gradings at 4, 5 and 6 months. A sensory panel found that most flavour and aroma attributes and maturity were in the order of R8>P8>R1=P1. The results of this study suggest that NSLAB play an important role in the development of flavour in Cheddar cheese by contributing to the production of amino acids and fatty acids.     

Influence of Homofermentative Lactobacilli on Physicochemical and Sensory Properties of Cheddar Cheese

Cheddar cheeses were produced under pilot plant conditions using a commercial Streptococcus culture amended with one of 10 homofermentative Lactobacillus strains. During the ripening period, pH, acidity, salt, moisture, fat, texture, fissure formation, gas development and sensory status were evaluated. Lactobacillus treated cheese did not differ much from the control in pH and acidity but acidity increased substantially after draining and cheddaring. Lactobacillus numbers increased at all stages as compared with the uninoculated control. High quality Cheddar cheese was produced by L. casei-subsp-casei (119-10/62) and L. casei-subsp-pseudoplantarum (137-10/62) from 7 to 12 vats aged for 2 months at 15°C and for a further 10 months at 7°C or 15°C. Fissure formation was observed in cheese made with L. casei-subsp-rhamnosus, one of the four cultures of L. casei-subsp-casei (LH13) and two of the three strains of L. casei-subsp-pseudoplantarum (83-4-12/62 and L3E). Certain Lactobacillus strains produced cheese with slight flavor defects. Other strains, in particular L. casei-subsp-rhamnosus, contributed to high acidity (72 - 0.89° domic) and low pH (5.2) at salting.     

Survival of micro‐organisms and organic acid profile of probiotic Cheddar cheese from buffalo milk during accelerated ripening

The study aimed to assess the impact of ripening at elevated temperatures on the survival of probiotic micro‐organisms and production of organic acids in Cheddar cheese. Cheese was manufactured from buffalo milk using lactococci starters along with different probiotic bacteria (Lactobacillus acidophilus LA‐5, Bifidobacterium bifidum Bb‐11 and Bifidobacterium longum BB536) as adjunct cultures. The cheeses were ripened at 4–6 °C or 12–14 °C for 180 days and examined for composition, organic acids and microbial survival. The production of organic acids was accelerated at 12–14 °C when compared to normal ripening temperatures. The probiotic bacteria increased production of lactic and acetic acids, compared to cheese made with lactococci alone. The survival of the mesophilic starters was significantly (P < 0.05) reduced in all the cheese samples ripened at the higher temperature. However, the probiotic bacteria remained viable (>7.0 log₁₀ cfu/g) throughout the 180 days of ripening, irrespective of temperature. It was concluded that Cheddar containing additional probiotic cultures can effectively be ripened at elevated temperatures without any adverse effects.     

Effect of Free or Encapsulated Recombinant Aminopeptidase of Lactobacillus rhamnosus S93 on Acceleration of Cheddar Cheese Ripening

The use of recombinant aminopeptidase (PepN) from Lactobacillus rhamnosus S93 in free or encapsulated form was investigated to shorten the duration of Cheddar cheese ripening. Proteolysis was determined by measuring the soluble nitrogen as phosphotungstic acid (PTA-N) derivatives and free amino acids (FAA) over a 6-month period. The experimental cheeses received higher scores for sensory properties than the control cheese. The amounts of PTA-N and total FAA in the cheese with the encapsulated enzyme after 2 months of ripening were close to those of the control cheese after 6 months, suggesting the acceleration in proteolysis by about 4 months.     

Dynamic Rheological Properties of Process Cheese: Effect of Ca and P Content,Residual Lactose,Salt-to-Moisture Ratio and Cheese Temperature

Four treatments of Cheddar cheese with two levels (high and low) of calcium (Ca) and phosphorus (P), and two levels (high and low) of residual lactose were manufactured. Each treatment was subsequently split prior to the salting step of cheese manufacturing process and salted at two levels (high and low) for a total of eight treatments. After two months of ripening, each treatment of Cheddar cheese was used to manufacture process cheese using a twin-screw Blentech process cheese cooker. NFDM, butter oil, trisodium citrate (emulsifying salt), and water were added along with Cheddar cheese for process cheese formulation. All process cheese food formulations were balanced for moisture (43.5%), fat (25%), and salt (2%), respectively. Dynamic rheological characteristics (G′ and G″) of process cheese were determined at 1.5Hz frequency and 750 Pa stress level by using a Viscoanalyzer during heating and cooling, temperature ranges from 30°C to 70°C then back to 30°C. High Ca and P content, and high S/M (HHH and HLH) cheeses had the significantly higher elastic (G′) and viscous (G″) modulus than other cheeses during heating from 30°C to 70°C, and cooling from 70°C to 30°C. No significant difference was observed among the other process cheeses during heating and cooling. Viscoelastic properties of process cheeses were also determined in terms of transition temperature (where G′?=?G″), and tan δ during heating (30°C to 70°C). Cheeses with high Ca and P, high lactose, and high S/M content had higher transition temperature than low Ca and P, low lactose, and low S/M content process cheeses. Low Ca and P and low S/M content cheeses (LLL, LHH, LHL, HLL) exhibited more viscous characteristics than high Ca and P and high S/M content process cheeses (HHL, HLH, LLH, HHH) during heating from 30°C to 70°C. Low Ca and P, low lactose, low S/M content (LLL) process cheese was observed for highest tan δ values (0.39 to 1.43), whereas high Ca and P, high lactose, high S/M content process (HHH) had the least (0.33 to 1.06) during heating. This study demonstrates that different characteristics of natural cheese used in process cheese manufacturing have significant impact on process cheese rheological and viscoelastic properties.     

Use of high pressure treatment to attenuate starter bacteria for use as adjuncts for Cheddar cheese manufacture

Attenuated starter bacteria cannot produce acid during cheese manufacture, but contain enzymes that contribute to cheese ripening. The aim of this study was to investigate attenuation of starter bacteria using high pressure treatment, for use in combination with a primary starter for Cheddar cheese manufacture, and to determine the effect of such adjunct cultures on secondary proteolysis during ripening. Lactococcus lactis ssp. cremoris HP and L. lactis ssp. cremoris 303 were attenuated by pressure treatment at 200 MPa for 20 min at 20 °C. Cheddar cheese was manufactured using untreated cultures of both these starter strains, either alone or in combination with their high pressure-treated equivalents. High pressure-treated starters did not produce acid during cheese manufacture and starter counts in cheeses manufactured using high pressure-treated starter did not differ from those of the controls. Higher levels of cell lysis were apparent in cheese manufactured using high pressure-treated strains than in the controls after 26 d of ripening. Small differences were observed in the peptide profiles of cheeses, analysed by reversed-phase HPLC; cheeses manufactured using high pressure-treated starters also had slightly higher levels of amino acids than the relevant controls. Overall, addition of high pressure-treated starter bacteria as a secondary starter culture accelerated secondary proteolysis in Cheddar cheese.

Industrial relevance

Attenuated starters provide extra pool of enzymes, which can influence cheese ripening, without affecting the cheese making schedule. This paper presents an alternative method for attenuation of starter bacteria using high pressure treatment and their subsequent use to accelerate secondary proteolysis in Cheddar cheese during ripening.     

Accelerated ripening of Cheddar cheese at elevated temperatures

Blocks (20 kg) of Cheddar cheese from a single vat were obtained from a local factory. Half the cheeses were cooled rapidly (15 h) to ripening temperature (8, 12 or 16 °C) and half were cooled slowly over 8 days to the same ripening temperatures. Cheeses were ripened for 9 months at 7 different time/temperature combinations. Ripening temperature had little influence on the number of non-starter lactic acid bacteria in the cheeses after 9 months, although rapid cooling to and ripening at 8 °C drastically reduced the growth rate of these adventitious bacteria. Proteolysis (as determined by urea-polyacrylamide gel electrophoresis; increases in water-soluble N; increases in phosphotungstic acid-soluble N; Cd ninhydrin-reactive amino groups; and reverse-phase HPLC) and lipolysis were accelerated by increasing the ripening temperature and by slow cooling of the cheeses. The rate of ripening was increased or decreased by changing the temperature. Cheeses ripened at 16 °C generally received the highest flavour scores, particularly early during ripening. However, the texture of these cheeses deteriorated after prolonged ripening at 16 °C. Maturation at 12 °C was considered to be optimal for the commercial acceleration of Cheddar cheese ripening.     

Proteolysis of Mahon cheese as affected by acoustic-assisted brining

 Mahon cheeses were brined in the presence of an ultrasonic field and ripened during 75 days at 12  °C and 85% RH. Secondary proteolysis (water-soluble N, non-protein N, and free amino acids) was measured and compared to that obtained for cheeses conventionally brined. There were no differences in water-soluble and non-protein N attributable to the brining treatment. However, cheeses acoustically brined exhibited higher concentrations of free amino acids. The release of total free amino acids was more pronounced during the first 15 days of ripening for both types of brining treatments. The changes in proteolysis (free amino acids) during cheese ripening caused by acoustic-assisted brining are indicative of a higher extent of proteolysis and may also improve cheese flavor. Received: 13 March 2000     

The effect of application of cold natural smoke on the ripening of Cheddar cheese

The present study was undertaken to study the effects of application of natural wood smoke on ripening of Cheddar cheese, and to determine the effects of smoking before or after ripening on cheese quality. A 20-kg block of Cheddar cheese obtained immediately after pressing was divided into six approximately 3-kg blocks and ripened at 8 degrees C for up to 270 d. One 3-kg block was taken after 1 d, 1, 3, 6, or 9 mo and smoked for 20 min, then returned to the ripening room for further ripening. Cheeses were sampled at intervals for lactobacilli counts, moisture, pH, and proteolysis. Sensory analysis was conducted on 6 and 9-mo-old cheeses by a trained sensory panel (n = 7). Results show that application of natural wood smoke did not significantly affect cheese pH or primary proteolysis during ripening. However, secondary proteolysis as assessed by the concentrations of free amino acids was generally higher in smoked cheeses than in control cheeses after 6 mo of ripening. Cheese smoked after 6 mo of ripening had better smoked flavor than that smoked after 9 mo of ripening. Cheese smoked after 3 mo of age and further ripened for 6 mo had the highest smoked flavor intensity. It is concluded that it is best to smoke cheese after ripening for at least 3 mo.     

Proteolytic pattern and organic acid profiles of probiotic Cheddar cheese as influenced by probiotic strains of Lactobacillus acidophilus,Lb. paracasei,Lb. casei or Bifidobacterium sp.

Cheddar cheeses were produced with starter lactococci and Bifidobacterium longum 1941, B. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. paracasei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilus LAFTI^® L10 to study the survival of the probiotic bacteria and the influence of these organisms on proteolytic patterns and production of organic acid during ripening period of 6 months at 4 °C. All probiotic adjuncts survived the manufacturing process of Cheddar cheese at high levels without alteration to the cheese-making process. After 6 months of ripening, cheeses maintained the level of probiotic organisms at >8.0 log₁₀ cfu g⁻¹ with minimal effect on moisture, fat, protein and salt content. Acetic acid concentration was higher in cheeses with B. longum 1941, B. lactis LAFTI^® B94, Lb. casei 279 and Lb. paracasei LAFTI^® L26. Each probiotic organism influenced the proteolytic pattern of Cheddar cheese in different ways. Lb. casei 279 and Lb. paracasei LAFTI^® L26 showed higher hydrolysis of casein. Higher concentrations of free amino acids (FAAs) were found in all probiotic cheeses. Although Bifidobacterium sp. was found to be weakly proteolytic, cheeses with the addition of those strains had highest concentration of FAAs. These data thus suggested that Lb. acidophilus 4962, Lb. casei 279, B. longum 1941, Lb. acidophilus LAFTI^® L10, Lb. paracasei LAFTI^® L26 and B. lactis LAFTI^® B94 can be applied successfully in Cheddar cheese.     

Characterization of aromatic properties of old-style cheese starters

Old-style cheese starters were evaluated to determine their ability to produce cheese aroma compounds. Detailed analyses of the aroma-producing potential of 13 old-style starter cultures were undertaken. The proteolytic profile of the starters was established by an accelerated ripening study using a model cheese slurry and compared with those of a commercial aromatic starter and commercial Cheddar cheeses. To evaluate the aromatic potential of the starter cultures, quantification of free amino acids liberated and volatile compounds after 15 d of ripening at 30°C as well as sensory analysis were carried out. Results showed that proteolysis patterns of all 13 starter cultures in the curd model were comparable to those of commercial Cheddar cheeses. All tested cultures demonstrated the ability to produce high amounts of amino acids recognized as precursors of aroma compounds. Several differences were observed between the starters and commercial Cheddar cheeses regarding some amino acids such as glutamate, leucine, phenylalanine, proline, and ornithine, reflecting the various enzymatic systems present in the starters. Starters Bt (control) and ULAAC-E exhibited various significant differences regarding their free amino acid profiles, as confirmed by sensory analysis. In addition, identification of volatile compounds confirmed the presence of several key molecules related to aroma, such as 3-methylbutanal and diacetyl. Besides the aroma-producing aspect, 2 starters (ULAAC-A and ULAAC-H) seem to possess an important ability to generate large amounts of γ-aminobutyric acid, which contributed up to 15% of the total amino acids present in the model curd after 15 d ripening. γ-Aminobutyric acid is an amine well-known for its antihypertensive and calming effects.     

The effect of ripening and cooking temperatures on proteolysis and lipolysis in Manchego cheese

The influence of ripening temperature on proteolysis and lipolysis was studied on four lots of raw ewe's milk Manchego cheese held for 60 days at 5, 10, 15 or 20°C. Mean levels of pH 4·6, trichloroacetic acid and phosphotungstic acid soluble N in 20°C cheeses were 52%, 78% and 95% higher than the respective levels in 5°C cheeses at the end of the ripening period. Free fatty acids content after 60 days was 90% higher in 20°C cheeses than in 5°C cheeses. Significant effects of the cooking temperature of the curd (30, 36, 38 or 40°C) on pH, moisture and NaCl content were recorded, but levels of nitrogenous fractions or free fatty acids in 60-day cheeses were not affected.     

Ripening of Cheddar cheese made from blends of raw and pasteurised milk

Cheddar cheeses were made from pasteurised milk (P), raw milk (R) or pasteurised milk to which 10 (PR10), 5 (PR5) or 1 (PR1) % of raw milk had been added. Non-starter lactic acid bacteria (NSLAB) were not detectable in P cheese in the first month of ripening, at which stage PR1, PR5, PR10 and R cheeses had 10⁴, 10⁵, 10⁶ and 10⁷ cfu NSLAB g⁻¹, respectively. After ripening for 4 months, the number of NSLAB was 1–2 log cycles lower in P cheese than in all other cheeses. Urea–polyacrylamide gel electrophoretograms of water-soluble and insoluble fractions of cheeses and reverse-phase HPLC chromatograms of 70% (v/v) ethanol-soluble as well as -insoluble fractions of WSF were essentially similar in all cheeses. The concentration of amino acids were pro rata the number of NSLAB and were the highest in R cheese and the lowest in P cheese throughout ripening. Free fatty acids and most of the fatty acid esters in 4-month old cheeses were higher in PR1, PR5, PR10 and R cheeses than in P cheese. Commercial graders awarded the highest flavour scores to 4-month-old PR1 cheeses and the lowest to P or R cheese. An expert panel of sensory assessors awarded increasingly higher scores for fruity/sweet and pungent aroma as the level of raw milk increased. The trend for aroma intensity and perceived maturity was R>PR10>PP5>PR1>P. The NSLAB from raw milk appeared to influence the ripening and quality of Cheddar cheese.