Autolysis of selected Lactobacillus helveticus adjunct strains during Cheddar cheese ripening

Cheddar cheeses were manufactured on a pilot scale (500 L vats) with three different Lactobacillus helveticus strains, which showed varying degrees of autolysis, added as adjuncts to the starter. Autolysis of adjunct strains was monitored by reduction in cell numbers, level of intracellular enzymes released into the cheese, and by the consequent changes in the degree of proteolysis and concentration of free amino acids in the cheese. The flavour profiles of the cheeses at 6 months were also determined. Significant variation in viability of the Lb. helveticus strains, which showed a positive correlation with the indicators of autolysis, was observed. However, cheese manufactured with the most autolytic strain did not receive the highest flavour scores. The results indicate that whereas autolysis of adjunct strains is an important factor in Cheddar cheese flavour development, other factors also contribute to the overall flavour improvement observed.     

Comparative analysis of the autolytic potential of Lactobacillus helveticus strains during Cheddar cheese ripening

Phenotypic tests and species-specific polymerase chain reaction (PCR) were used to confirm the classification of 41 isolates of Lactobacillus helveticus obtained from different sources. Randomly amplified polymorphic DNA–PCR (RAPD–PCR) was applied to the differentiation of these isolates, which grouped into nine clusters at the 80% similarity level. To determine if the different clusters had different degrees of autolysis, a representative selection of 14 isolates from different clusters was selected and screened for autolytic characteristics in vitro and also in Cheddar cheesemaking trials. No correlation could be found between the cluster from which the isolate originated, the levels of autolysis in the cheese, or the results of the in vitro screening. It was concluded that cheese manufacture per se is the best method to determine the potential for and the degree of autolysis during cheesemaking for a given isolate.     

Early lysis of Lactobacillus helveticus CNRZ 303 in Swiss cheese is not prophage-related

Lactobacillus helveticus is mainly used as starter in Swiss-type cheeses. Often, lysogenic strains are eliminated because of the risk of early lysis and acidification failure due to phage expression. On the other hand, L. helveticus lysis was shown to positively influence cheese proteolysis during ripening. In order to better assess the relationship between lysis and lysogeny, a prophage-cured derivative of L. helveticus CNRZ 303 was isolated (LH 303-G11) and relysogenised (LH 303-G11R), as demonstrated by hybridisation using the whole phage DNA as probe. The growth, lysis in buffered solutions and lytic activities in zymogram using either Micrococcus luteus or L. helveticus as substrate were identical between the mother strain and its cured derivatives. Only morphological differences were observed by scanning electron microscopy: the cells of the cured derivative were shorter in length. The mother strain and its cured and relysogenised derivatives were assayed in triplicate in experimental Swiss cheeses (scale 1:100). No differences were noted during the cheese making: the three strains exhibited identical kinetics of acidification, leading to similar cheeses at day 1 in terms of gross composition and pH. Phages were detected only in the cheeses made with the mother strain and the relysogenised derivative. The lysis of L. helveticus, estimated by viability decrease and release of the intracellular marker D-lactate deshydrogenase, started early before brining and continued during the cold room ripening. No obvious differences of lysis extent were observed. These results demonstrated for the first time that, in the case of LH 303, the extensive lysis observed in cheese is mainly due to autolysin activity and not to prophage induction.     

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.     

Variation in caseinolytic properties of six cheese related Lactobacillus helveticus strains

A large degree of strain variation was observed in caseinolytic properties of six cheese related Lactobacillus helveticus strains. Activity on intact α_s1- and β-casein was observed only after growth in milk and not in MRS. Totally 27 peptides from α_s1- and 22 from β-casein were identified from MS/MS fragmentation patterns. All six strains released peptides from the amino end of α_s1-casein, and the bonds Ile₆-Lys₇ and Gln₉-Gly₁₀ were identified as primary cleavage sites. Strain variation in the activity on intact β-casein was observed and five of the six strains released peptides from the C-terminal region. The strains had very different activities and some strains had only trace activities. L. helveticus CNRZ 32 had the highest activity towards α_s1-casein while L. helveticus LHC2 had the highest activity towards β-casein, and these two strains also produced unique peptides from both α_s1- and β-casein.     

Autolysis and related proteolysis in Swiss cheese for two Lactobacillus helveticus strains

Intracellular peptidases of Lactobacillus helveticus may play a major role in the proteolysis of Swiss cheeses, provided that they are released through bacterial lysis. Experimental Swiss cheeses were manufactured on a small scale from thermized and microfiltered milk using as starters (in addition to Streptococcus thermophilus and Propionibacterium freudenreichii) one of two Lb. helveticus strains, ITGLH1 and ITGLH77, which undergo lysis to different extents in vitro. All the cheeses were biochemically identical after pressing. The viability of Lb. helveticus ITGLH1 and ITGLH77 decreased to a similar extent (96-98%) while in the cold room, but the concomitant release of intracellular lactate dehydrogenase in cheeses made with strain ITGLH1 was 5-7-fold that in cheeses made with ITGLH77. Protein profiles and immunoblot detection of the dipeptidase PepD confirmed a greater degree of lysis of the ITGLH1 strain. Free active peptidases were detected in aqueous extracts of cheese for both strains, and proteolysis occurred principally in the warm room. Reversed-phase HPLC revealed a more extensive peptide hydrolysis for ITGLH1, which was confirmed by the greater release of free NH2 groups (+33%) and free amino acids (+75%) compared with ITGLH77. As the intracellular peptidase activities of ITGLH1 and ITGLH77 have previously been shown to be similar, our results indicated that the extent of lysis of Lb. helveticus could have a direct impact on the degree of proteolysis in Swiss cheeses.     

Variation in aminopeptidase and aminotransferase activities of six cheese related Lactobacillus helveticus strains

Aminopeptidase and aminotransferase activities of six Lactobacillus helveticus strains grown in milk and in a peptide rich glucose based medium de Man Rogosa Sharpe (MRS) were examined. The strains had similar specificity for ten aminopeptidase substrates but some significant (P < 0.05) strain differences in activity levels. Higher aminopeptidase activity was found after growth in milk against almost all substrates. Aminotransferase activity was detected against 13 amino acids. All strains had highest activity against aromatic amino acids followed by branched-chain amino acids. Five strains had activity against Met. Activity against Asn, Asp, Lys, Gln, His and Pro was detected among four strains but only after growth in MRS. Significantly (P < 0.05) higher activity against aromatic amino acids was found after growth in MRS. No obvious differences were found in activities against branched-chain amino acids and Met because of growth medium. These results may offer potential to choose strains that accelerate amino acid release in cheese with or without introducing new aroma notes during ripening.     

Flavour-enhancement of low-fat Kashkaval cheese using heat-or freeze-shocked Lactobacillus delbrueckii var. helveticus cultures

Kashkaval cheese of different fat contents was manufactured using heat- or freeze-shocked cultures of Lactobacillus delbrueckii var. helveticus added at a level of 2% to cheese milk prior to renneting. Levels of moisture, total N, salt or titratable acidity of cheeses with different fat levels were different. Proteolysis and lipolysis in low-fat Kashkaval cheese without additives were lower than those found in full-fat cheese. Incorporation of heat- or freeze-shocked L. delbrueckii var. helveticus cultures into milk of low-fat cheese greatly enhanced proteolysis and increased slightly the levels of free fatty acids. Regular low fat cheese did not develop adequate Kashkaval cheese flavour and the cheese was firm. However, addition of heat- or freeze-shocked cultures increased the flavour intensity and improved body and texture of the resultant cheese, yielding low-fat cheese with flavour intensity and body characteristics similar to those of standard fat cheese at each stage of ripening.     

Acid phosphatases activity in cheese and starters.

The acid phosphatase activity levels in a number of Greek cheeses and in Cheddar cheeses were found to be unaffected by storage for up to 18 months and 12 months respectively. In Cheddar cheese, starter organisms made an insignificant contribution to this activity. Studies of acid phosphatase prepared from Streptococcus cremoris-lactis NCDO 762 starter cultures showed that the enzyme was of high molecular weight and largely particle-bound. The pH of optimum activity was 5-2 and the enzyme was inhibited by F-minus,Al-3+, a number of heavy metals, oxidizing agents and sulphydryl-modifying reagents. Kinetic measurements at pH 5-2 gave a Km value for p-nitrophenyl phosphate of 1-2 mM. Orthophosphate, pyrophosphate and isoelectrically precipitated casein behaved as competitive inhibitors to the hydrolysis of p-nitrophenyl phosphate with Ki values of 1-2 mM, 1-0 mM, 1-0 MM and 1-1 mM respectively. In spite of this binding to the enzyme, casein provided a very poor substrate for the starter acid phosphatase. The properties of acid phosphatase present in Cheddar cheese made with Str. cremoris NCDO 924 starter were consistent with the enzyme being exclusively of milk origin and small differences between this and the acid phosphatase previously isolated from bovine milk were attributable to the binding of peptides produced during the cheese maturation to the enzyme molecules. It was concluded that in cheese, phosphatase action was due largely to the enzyme of milk origin, with that provided by the starter being of minor importance.     

Slower proteolysis in Cheddar cheese made from high-protein cheese milk is due to an elevated whey protein content

A growing number of companies within the cheese-making industry are now using high-protein (e.g., 4–5%) milks to increase cheese yield. Previous studies have suggested that cheeses made from high-protein (both casein and whey protein; WP) milks may ripen more slowly; one suggested explanation is inhibition of residual rennet activity due to elevated WP levels. We explored the use of microfiltration (MF) to concentrate milk for cheese-making, as that would allow us to concentrate the casein while varying the WP content. Our objective was to determine if reducing the level of WP in concentrated cheese milk had any impact on cheese characteristics, including ripening, texture, and nutritional profile. Three types of 5% casein standardized and pasteurized cheese milks were prepared that had various casein:true protein (CN:TP) ratios: (a) control with CN:TP 83:100, (b) 35% WP reduced, 89:100 CN:TP, and (c) 70% WP reduced, 95:100 CN:TP. Standardized milks were preacidified to pH 6.2 with dilute lactic acid during cheese-making. Composition, proteolysis, textural, rheological, and sensory properties of cheeses were monitored over a 9-mo ripening period. The lactose, total solids, total protein, and WP contents in the 5% casein concentrated milks were reduced with increasing levels of WP removal. All milks had similar casein and total calcium levels. Cheeses had similar compositions, but, as expected, lower WP levels were observed in the cheeses where WP depletion by MF was performed on the cheese milks. Cheese yield and nitrogen recoveries were highest in cheese made with the 95:100 CN:TP milk. These enhanced recoveries were due to the higher fraction of nitrogen being casein-based solids. Microfiltration depletion of WP did not affect pH, sensory attributes, or insoluble calcium content of cheese. Proteolysis (the amount of pH 4.6 soluble nitrogen) was lower in control cheeses compared with WP-reduced cheeses. During ripening, the hardness values and the temperature of the crossover point, an indicator of the melting point of the cheese, were higher in the control cheese. It was thus likely that the higher residual WP content in the control cheese inhibited proteolysis during ripening, and the lower breakdown rate resulted in its higher hardness and melting point. There were no major differences in the concentrations of key nutrients with this WP depletion method. Cheese milk concentration by MF provides the benefit of more typical ripening rates.     

An application in Gouda cheese manufacture for a strain of Lactobacillus helveticus ND01

Gouda cheese was manufactured with Lactococcus lactis ssp. lactis IMAU60010, L. lactis ssp. cremoris IMAU40136 and L. helveticus ND01 isolated from the naturally fermented milk in China. Starter cultures added with L. helveticus ND01 produced Gouda cheese with dramatically more proteolysis than control cheeses. Compared with control cheese, experimental cheese with L. helveticus ND01 adjunct revealed dramatic increase in both Angiotensin I‐converting enzyme (ACE)‐inhibitory activity and γ‐aminobutyric acid content. The ACE‐inhibitory activity of Gouda cheeses with the addition of 1 × 10⁵ CFU/mL L. helveticus ND01 increases from 53.7 to 83.1% at 6 weeks of ripening.     

Effect of commercial lipolytic enzymes on flavour development in Cheddar cheese

The addition of commercial lipolytic enzymes to experimental Cheddar cheese accelerated the liberation of free fatty acids during ripening. The substrate specificity of the added enzymes generally governed the chain lengths of the free fatty acids in the cheeses. None of the enzymes accelerated the formation of typical flavour in either the presence or the absence of a flavour-enhancing proteinase, but higher addition levels produced lipolytic rancidity.     

Effect of Lactobacillus helveticus and Propionibacterium freudenrichii ssp. shermanii combinations on propensity for split defect in Swiss cheese

One of the least controlled defects in Swiss cheese is development of splits that appear during refrigerated storage after cheese is removed from the warm room. Such fissures, or cracks, in the body of the cheese can be as short as 1 cm, or long enough to span a 90-kg block. A 2 x 2 x 2 factorial experiment was used to determine the effect of different Lactobacillus helveticus/Propionibacterium freudenreichii ssp. shermanii starter culture combinations on the occurrence of split defect in Swiss cheese. Eights vats of cheese were made in summer and eight in winter. Each 90-kg block of cheese was cut into twenty-four 4-kg blocks and graded based on the presence of splits. Only small variations were found in the composition of cheeses made during the same season. There were no correlations between moisture, pH, fat, protein, calcium, lactose contents, D/L lactate ratio, or protein degradation that could be used to predict splits after 90 d of storage. However, cheese made in the summer had 2% higher moisture content and a greater prevalence of splits. There was a sixfold increase in amount of downgraded cheese between the best and worst culture combinations used during cheese manufacture. After 90-d storage, 14 to 90% of cheese had splits in the summer, and 1 to 6% in the winter. Split formation increased with time from 60 to 120 d of storage and extent of split formation was influenced by both the lactobacilli and propionibacteria cultures used.     

Manufacture of low-fat Cheddar cheese by exopolysaccharide-producing Lactobacillus plantarum JLK0142 and its functional properties

This study aimed to evaluate the effect of exopolysaccharide (EPS)-producing Lactobacillus plantarum JLK0142 on the ripening characteristics and in vitro health-promoting benefits of low-fat Cheddar cheese. Three batches of cheese were made by employing a non-EPS–producing cheese starter (control), in combination with Lb. plantarum JLK0142 as an adjunct and the purified EPS as an ingredient. Lactobacillus plantarum JLK0142 survived well in cheese, with counts of 7.99 log cfu/g after 90 d of ripening. All experimental cheeses (with adjunct culture or EPS ingredient) had higher moisture, proteolysis, and sensory scores, and lower hardness and cohesiveness compared with the control cheese. Water-soluble extracts from the experimental cheeses outperformed that of the control in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and hydroxyl radicals, and inhibiting α-amylase, angiotensin-converting enzyme, and HT-29 tumor cell growth. Therefore, incorporation of the EPS-producing culture of Lb. plantarum JLK0142 is promising for improvement of low-fat cheese quality and bioactivities.     

瑞士乳杆菌对契达干酪成熟期间所产ACE抑制肽的影响及其消化稳定性

为明确瑞士乳杆菌对契达干酪中血管紧张素转换酶(angiotensin-converting enzyme,ACE)抑制肽活性的影响,以蛋白质水解度和ACE抑制率为指标,与干酪乳杆菌组、鼠李糖乳杆菌组和空白组干酪进行对照,研究瑞士乳杆菌对干酪成熟期间蛋白质水解及ACE抑制活性的影响,并对ACE抑制活性最高时期的干酪进行消...     

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.     

Application of Lactobacillus amylovorus as an antifungal adjunct to extend the shelf-life of Cheddar cheese

Lactobacillus amylovorus DSM 19280 is an antifungal strain that is inhibitory to a range of fungi including Penicillium expansum, Penicillium roqueforti, Aspergillus niger, Aspergillus fumigatus and Fusarium culmorum. In this study, the strain was used as an adjunct culture in a Cheddar cheese model system. During the ripening period, P. expansum spores were applied to the cheese surface to mimic fungal contamination. The presence of the antifungal L. amylovorus adjunct resulted in a four-day delay in appearance of Penicillium growth on the cheese in comparison to the adjunct-free control. When cheeses were exposed to natural airborne fungi, the presence of the adjunct resulted in a six-day delay in the appearance of mycelia on the cheese surface. Significantly, its presence had no detectable negative impact on cheese quality. The results indicate that the strain could have an application for extending the shelf-life of cheeses which are prone to fungal spoilage.     

The effect of some manufacturing conditions on the development of flavour in Cheddar cheese

Organoleptic assessments by the NIRD panel of Cheddar cheeses made with Streptococcus cremoris NCDO 924 or 1986, either in enclosed vats excluding nonstarter flora or in open vats, showed that high viable starter populations in curd did not give stronger-flavoured cheese, but led to the development of bitterness. Cheeses made in open vats developed typical flavour more rapidly than those made in enclosed vats. Maturation temperature was the most important factor in determining the flavour intensity; cheese ripened at 13d?C for six months had stronger flavour than corresponding ones ripened at 6d?C for nine months, irrespective of the starter or vat used.     

干酪成熟过程中发酵剂的作用

介绍干酪成熟过程中乳酸菌发酵剂及二级发酵剂的作用,包括在干酪风味、质地、加速干酪成熟、产生抗菌素及营养和颜色等多方面作用,同时分析成熟期间发酵剂控制不当会引起的负面作用,提出应用具有选择性的发酵剂或经遗传修饰的发酵剂生产干酪具有较强的应用前景。