Probiotic Cheddar cheese: Influence of ripening temperatures on survival of probiotic microorganisms, cheese composition and organic acid profiles

Bifidobacterium longum 1941, Bifidobacterium animalis subsp. lactis LAFTI^®B94 (B94), Lactobacillus casei 279, Lb. casei LAFTI^®L26 (L26), Lactobacillus acidophilus 4962 or Lb. acidophilus LAFTI^®L10 (L10) were used as an adjunct in the production of Cheddar cheeses which were ripened for 24 wk at 4 and 8 °C. Effects of ripening temperatures on survival of starter lactococci and probiotic microorganisms, pH and composition of cheeses and production of organic acids were examined. The counts of starter lactococci in cheeses produced with B. animalis B94, Lb. casei L26 or Lb. acidophilus 4962 ripened at 8 °C were significantly lower than those ripened at 4 °C (P < 0.05) at 24 wk. Probiotic microorganisms remained viable (>7.50 log₁₀ CFU/g) at the end of 24 wk and their viability was not affected by the ripening temperatures. There were significant effects of the type of probiotic microorganisms used, ripening time, ripening temperatures and their interactions on the concentration of lactic and acetic acids in the cheeses (P < 0.05). The acetic acid concentration in cheeses made with Bifidobacterium sp. or Lb. casei sp. was significantly higher than that of the control cheese (P < 0.05). Citric, propionic and succinic acids contents of the cheeses were not significantly affected by the type of probiotic microorganisms or ripening temperatures (P > 0.05).     

Isomaltooligosaccharide increases the Lactobacillus rhamnosus viable count in Cheddar cheese

Five batches of Cheddar cheese were manufactured containing different levels of isomaltooligosaccharide (IMO) and a probiotic strain of Lactobacillus rhamnosus to study the effect of IMO on the survival of starter lactococci and probiotic micro‐organisms, on proteolytic patterns, cheese composition and sensory properties. The cheese was exposed to conditions simulating those found in the gastrointestinal tract to evaluate the survival of Lb. rhamnosus. Results demonstrated that the addition of Lb. rhamnosus and IMO did not affect the main compositional variables of Cheddar cheese. The counts of starter culture and probiotic organisms increased in cheese which contained Isomaltooligosaccharide (Batches 3, 4 and 5) more than in the control (Batches 1 and 2) during the fermentation. The probiotic counts in fresh cheese (B‐4) was 9.23 log₁₀ cfu/g which was more than one log cycle greater than in the control (B‐2). The probiotic counts remained above 8 log₁₀ cfu/g at the end of the manufacturing process. Primary proteolysis was not affected by the addition of probiotic bacteria and IMO, but the level of secondary proteolysis was slightly higher compared with the control group. The addition of IMO improved the texture and sensory quality of the cheese and the probiotic bacterium had the same effect. Under conditions that simulated the gastrointestinal tract, the probiotic bacteria in cheese (B‐4) exhibited good survival and remained above the recommended 6–7 log₁₀ cfu/g.     

Preliminary screening of Bifidobacteria spp. and Pediococcus acidilactici in a Swiss cheese curd slurry model system: Impact on microbial viability and flavor characteristics

A Swiss cheese curd slurry model system was used as a preliminary screening method to determine the feasibility of the incorporation of probiotic bacteria (Bifidobacterium breve R0070, Bifidobacterium infantis R0033, Bifidobacterium longum R0175, and Pediococcus acidilactici R1001) into Swiss cheese. The cheese curd was inoculated with probiotic bacteria (8.0 log₁₀ cfu g⁻¹) and ripened anaerobically for 0, 7, and 10 days at 37 °C. Following ripening, counts of the probiotic bacteria increased to 9–10 log₁₀ cfu g⁻¹, with no significant difference in the viability of the four probiotic bacteria. Viable populations of Swiss cheese background microflora in the presence of each probiotic culture were comparable with the control. Ripening time, and to a lesser extent probiotic treatment had a significant effect on the content of several volatile flavor compounds. Similarly, ripening time contributed to a significant increase in the content of a majority of the free amino acids. The study demonstrated the feasibility of the incorporation of probiotic bacteria into Swiss cheese to produce a functional food.     

Evolution of Free Amino Acids during the Ripening of Cheddar Cheese Containing Added Lactobacilli Strains

Cheddar cheese was produced with different lactobacilli strains added to accelerate ripening. The concentration of proteolytic products was determined as free amino acids in the water-soluble fraction at two, four, seven and nine months of aging and at two different maturation temperatures (6°C, 15°C). All amino acids increased during ripening and were higher in the Lactobacillus- added cheeses than in the control cheese, and higher in cheeses ripened at 15°C than at 6°C. Glutamic acid, leucine, phenylalanine, valine and lysine were generally in higher proportion in all cheeses. The cheeses with added L. casei-casei L2A were classified as having a “strong Cheddar cheese” flavor after only seven months of ripening at 6°C.     

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.     

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.     

Probiotic Cheddar Cheese: Influence of Ripening Temperatures on Proteolysis and Sensory Characteristics of Cheddar Cheeses

ABSTRACT:  Bifidobacterium longum 1941, B. animalis subsp. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. casei LAFTI L26, Lb. acidophilus 4962, or Lb. acidophilus LAFTI L10 were used as an adjunct in the production of Cheddar cheeses, which were ripened at 4 and 8 °C for 24 wk. Effects of ripening temperatures and probiotic adjuncts on proteolysis and sensory evaluation of the cheeses were examined. Higher ripening temperature increased the level of proteolysis in the cheeses. Product of proteolysis and organic acids released during ripening were shown to be important for the flavor of Cheddar cheeses. There were positive and significant correlations between the levels of soluble nitrogen, lactic, acetic, and butyric acids, percentage hydrolysis of α_s1-CN and β-CN to the scores of cheddary flavor ( P < 0.05). Scores for sour-acid and vinegary flavors were higher in cheeses with the addition of Bifidobacterium sp. or Lb. casei 279 ripened at 8 °C. The scores were positively and significantly correlated to the level of lactic, acetic, and free amino acids in the cheeses ( P < 0.05). The results show that both 4 and 8 °C have potential for use in the ripening of probiotic Cheddar cheeses.     

Viability of the Lactobacillus rhamnosus HN001 Probiotic Strain in Swiss‐ and Dutch‐Type Cheese and Cheese‐Like Products

The objective of this study was to determine the viability of the probiotic Lactobacillus rhamnosus HN001 in Swiss‐type and Dutch‐type cheese and cheese‐like products (milk fat is substituted by stearin fraction of palm fat) during manufacture, ripening, and storage. The use of the probiotic L. rhamnosus HN001 in Dutch‐type cheese and cheese‐like products significantly (P = 0.1) changed their chemical composition (protein and fat content) and an insignificant increase (approximately 1.6% in cheese‐like products and approximately 0.3% in cheese) in yield. L. rhamnosus HN001 did not affect the rate of changes in the pH of ripened cheese and cheese‐like products. A minor increase in probiotic counts was observed in initial stages of production and were partially removed with whey. Ripened cheese and cheese‐like products were characterized by high survival rates of probiotic bacteria which exceeded 8 log CFU/g after ripening. An insignificant reduction in the number of viable probiotic cells was noted during storage of Swiss‐type and Dutch‐type cheese, whereas a significant increase in probiotic cell counts was observed in cheese‐like products during storage.     

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.     

Microbiological and physicochemical properties of dry‐cured neck inoculated with probiotic of Bifidobacterium animalis ssp. lactis BB‐12

The effect of inoculum level of Bifidobacterium animalis ssp. lactis BB‐12 probiotic strain and ripening period on the quality of dry‐cured neck was studied. The microbiological parameters (Enterobacteriaceae, LAB and TVC) and physicochemical attributes (pH value, a_w, TBARS index, colour) were determined directly after fermentation at 15 °C for 3 weeks, after 6 and 12 months of ripening at 4 °C. The highest LAB count and a lower pH value were found in the meat inoculated with probiotic strain at 6.6 log cfu g^?1 (B2) followed by inoculation with probiotic strain at 6.3 log cfu g^?1 (B1). Level of inoculation had not had an influence on water activity, TBARS index and total colour parameters. Changes of fat oxidation during half‐year of ripening were limited in probiotic meat samples compared to naturally fermented control meat (C). Based on the results, it can be concluded that the most favourable physicochemical and microbiological parameters of the dry‐cured neck were obtained after 6 months of ripening. At that time, the Bifidobacterium BB‐12 at both levels is a good potential starter for meat fermentation.     

Effects of Penicillium roqueforti and whey cheese on gross composition,microbiology and proteolysis of mould‐ripened Civil cheese during ripening

Four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as the secondary starter with and without addition of the whey cheese (Lor); in parallel, secondary starter‐free counterparts were manufactured. Chemical composition, microbiology and proteolysis were studied during the ripening. The incorporation of whey cheese in the manufacture of mould‐ripened Civil cheese altered the gross composition and adversely affected proteolysis in the cheeses. The inoculated P. roqueforti moulds appeared to grow slowly on those cheeses, and little proteolysis was evident in all cheese treatments during the first 90 days of ripening. However, sharp increases in the soluble nitrogen fractions were observed in all cheeses after 90 days. Microbiological analysis showed that the microbial counts in the cheeses were at high levels at the beginning of ripening, while their counts decreased approximately 1–2 log cfu/g towards the end of ripening.     

Bioactive action of β‐glucosidase enzyme of Bifidobacterium longum upon isoflavone glucosides present in soymilk

Bioconversion of isoflavone glucosides and antioxidant activity by probiotic strain (Bifidobacterium longum) during soymilk fermentation was investigated, as well as partial characterisation of the produced enzyme β‐glucosidase. The enzyme has higher affinity for genistin than for other substrates assayed. Maximum activity occurred at 42 °C and at pH 6.0; keeping 70–80% of activity for 60 days stored at low temperatures. Bifidobacterium longum grew well in soymilk (8.26 log CFU mL^?1 and pH of 3.9 at 24 h) and were produced in good quantities of organic acids. High hydrolysis degree of isoflavone glucosides (81.2%) was observed at 24 h. Enhancements in bioactivity were assessed in fermented soymilk by monitoring the radical‐scavenging activity, antioxidant activity and DNA protective action. The use of probiotic Bifidobacterium strain as β‐glucosidase producer increased bioactive isoflavone content and demonstrated that this enzyme plays a key role in the bioavailability of soymilk isoflavones, reducing the bioconversion time compared to other studies.     

The effect of three different ripening/storage conditions on the distribution of selected parameters in individual parts of Dutch‐type cheese

The aim of the study was (i) to detect changes of dry matter, NaCl and twenty‐two free amino acids contents, pH and levels of selected microorganisms in four layers of cheese (from edge to core) during ripening and storage period and (ii) to describe the changes of the above‐mentioned parameters caused by early relocation of cheese from optimum ripening conditions to refrigeration temperatures. The number of mesophilic aerobic and facultative anaerobic bacteria and lactic acid bacteria differed significantly (P < 0.05) during the experiment dependent on the analysed layer and ripening/storage conditions. The free amino acid content differed significantly in individual analysed layers of cheese and also according to individual ripening/storage conditions. The highest content of free amino acids was found in samples stored at optimal ripening temperatures. Cheese hardness was also analysed and the lowest one was detected in samples ripened under optimal temperatures for the whole period. Early release of cheeses into storage rooms with lower temperature significantly affected properties of these products.     

Effect of Penicillium roqueforti and incorporation of whey cheese on volatile profiles and sensory characteristics of mould‐ripened Civil cheese

In this study, four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as secondary starter for the manufacture of mould‐ripened Civil cheese with and without addition of the whey cheese Lor; in parallel, secondary starter‐free counterparts were manufactured. A total of 83 compounds were identified. Ketones, alcohols and esters were the principal classes of volatile components. Principal component analysis of the headspace volatiles grouped cheeses by age and type. P. roqueforti inoculated cheese was clearly separated from the other cheeses at 180 days of ripening, and these cheeses were characterised with high levels of ketones (e.g., 2‐butanone, 2‐heptanone). Differences in the panel scores between the cheese samples were not significant during the first stage of ripening (up to 60 days); as ripening proceeded, these differences were become evident and P. roqueforti inoculated cheeses received higher scores than others. Addition of Lor in the manufacture of mould‐ripened Civil cheese caused lower points by the sensory panel, and the cheese inoculated with P. roqueforti and Lor‐free was the best type of mould‐ripened Civil cheese. The results showed that the use of P. roqueforti in the manufacture of mould‐ripened Civil cheese has significant impact on the volatile profiles and sensory attributes.     

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.     

Identification of Lactic Acid Bacteria Isolated from Tulum Cheese During Ripening Period

The fresh Tulum cheese was manufactured and then ripened at 10 ± 2°C for 3 months. Isolation and identification of lactic acid bacteria (LAB) was carried out during the ripening period of 90 days. Lactic acid bacteria were isolated from PCA, M17, and MRS agar. The strains isolated were identified using morphology, colony pigmentation, production of carbon dioxide from glucose, growth at 4 and 40°C, salt tolerance, starch hydrolysis, and sugar fermentation with the API system methods. A total of 253 strains isolated during the storage. Identifying strains belonged to genera of the lactobacilli (133), pediococci (44), enterococci (29), leuconostocs (27), and lactococci (8). 12 of 253 strains were not also identified. Lactobacilli (75.2%) were frequently determined on MRS medium. In the results of this article, lactobacilli were found at high frequencies, while enterococci, lactococci, leuconostocs, and pediococci were found at low frequencies in Tulum cheese. Lactobacilli increased during the ripening period, but the others did not change a significant amount.     

Cream cheese as a symbiotic food carrier using Bifidobacterium animalis Bb‐12 and Lactobacillus acidophilus La‐5 and inulin

The stability of cream cheeses as a symbiotic food carrier, through supplementation with different concentrations of probiotic bacteria Bifidobacterium animalis Bb‐12 and Lactobacillus acidophilus La‐5 and the prebiotic ingredient inulin was investigated. Physicochemical parameters, pH values, total solids, fat and protein levels and the viable counts of the starter lactic culture Streptococcus thermophilus and probiotic cultures, were carried out at 1, 15, 30 and 45 days of refrigerated storage (8 ± 0.5 °C). Different physicochemical characteristics were observed in all formulations. S. thermophilus showed good viability in all the trials (6.66–9.38 log cfu/g), whereas B. animalis remained above 6 log cfu/g in all the trials during the period evaluated. However, L. acidophilus showed an accentuated decline, registering values of 3.1 log cfu/g at the end of the period studied. The results suggested that cream cheese was an adequate food matrix for supplementation with probiotic bacteria, in particular B. animalis, and the prebiotic ingredient, showing potential as a symbiotic food.     

Effects of encapsulation on the viability of probiotic strains exposed to lethal conditions

The effect of microencapsulation on the viability of Lactobacillus casei, L. paracasei, L. acidophilus Ki and Bifidobacterium animalis BB‐12 during exposure to lethal conditions (25% NaCl, pH 3.0 and 55–60 °C) was evaluated. Results demonstrated that survival of probiotic strains to the imposed lethal stress conditions was strain dependent. With the exception of exposure to 25% (w/v) NaCl, L. acidophilus Ki (free and encapsulated cells) demonstrated the highest survival rates through exposure to lethal conditions of temperature and pH. For this probiotic strain exposed to heat, microencapsulated cells expressed a higher heat tolerance at 55 °C than free cells. For the other tested bacteria, in general, encapsulation had no positive effect on survival through the tested lethal conditions.     

Nutritional Evaluation of Accelerated Ripened Cheddar Processed Cheese

Accelerated ripened Cheddar cheese was prepared by blending two parts of shredded curd made from standardized cow's milk with one part of 5.2% NaCl solution and ripening at 30°C for 8 days. On a dry matter basis, protein and fat of accelerated ripened cheese were similar to that of conventionally ripened Cheddar cheese, while lactose and total ash were greater. Similar observation was made for processed cheese samples. No change in vitamin A or in riboflavin but a fourfold increase in folic acid was observed during accelerated ripening. Protein efficiency ratio, net protein utilization and digestibility coefficient by rat tests were slightly but significantly higher for conventionally ripened processed cheese. However, no difference in biological value was observed.     

Release and identification of angiotensin-converting enzyme-inhibitory peptides as influenced by ripening temperatures and probiotic adjuncts in Cheddar cheeses

The aim of the study was to examine the release of angiotensin-converting enzyme (ACE)-inhibitory peptides in Cheddar cheeses made with starter lactococci and Bifidobacterium longum 1941, B. animalis subsp. lactis LAFTI^® B94, Lactobacillus casei 279, Lb. casei LAFTI^® L26, Lb. acidophilus 4962 or Lb. acidophilusLAFTI^® L10 during ripening at 4 and 8 °C for 24 weeks. ACE-inhibitory activity of the cheeses was maximum at 24 weeks. Cheeses made with the addition of Lb. casei 279, Lb. casei LAFTI^® L26 or Lb. acidophilus LAFTI^® L10 had significantly higher (P < 0.05) ACE-inhibitory activity than those without any probiotic adjunct after 24 weeks at 4 and 8 °C. The IC₅₀ of cheeses ripened at 4 °C was not significantly different (P > 0.05) to that ripened at 8 °C. The lowest value of the IC₅₀ (0.13 mg mL⁻¹) and therefore the highest ACE-inhibitory activity corresponded to the cheese with the addition of Lb. acidophilus LAFTI^® L10. Several ACE-inhibitory peptides were identified as κ-CN (f 96–102), α_s1-CN (f 1–9), α_s1-CN (f 1–7), α_s1-CN (f 1–6), α_s1-CN (f 24–32) and β-CN (f 193–209). Most of the ACE-inhibitory peptides accumulated at the early stage of ripening, and as proteolysis proceeded, some of the peptides were hydrolyzed into smaller peptides.