Substitution of natural whey starter by mixed strains of Lactobacillus helveticus in the production of Reggianito Argentino cheese

Reggianito Argentino cheeses were manufactured with mixed strains of Lactobacillus helveticus cultured in free viable bacteria whey. As controls, cheeses with natural whey starter were made. Gross composition of cheeses did not differ significantly. The number of total termophilic lactic acid bacteria at the end of ripening was near 10⁷ CFU/g, but when the strain Lh 209 was present in the mixture, this number was 10⁶ CFU/g. Soluble nitrogen at pH 4.6 did not differ between control and experimental cheeses, but soluble nitrogen in tricloroacetic acid 12% and phosphotungstic acid 2.5% showed significant differences at the end of ripening, being higher the values when the strain Lh 209 was present in the mixture. Electrophoretic profiles for control and experimental cheeses were very similar at 0, 90 and 180 days of ripening. An increase in the acid degree value of fat during ripening was noticeable indicating a lipolytic activity in the cheese matrix that was similar for the different strains mixtures. Results from sensory analysis did not show differences among the cheeses of these tests. Despite some differences in the production tricloroacetic and phosphotungstic acid soluble nitrogen were observed for mixtures in which Lh 209 was present, all cheeses were good quality Reggianito Argentino cheeses. These results open and interesting prospective for the use of selected strains culture in whey in substitution of natural whey.     

Production of peptides and free amino acids in a sterile extract describes peptidolysis in hard-cooked cheeses

Hard cooked cheeses are mostly manufactured with lactic starters of Lactobacillus helveticus, which constitute a major proteolytic agent in the food. In this work, we assessed the proteolysis produced by enzymes of two strains of L. helveticus in a new cheese model, which consisted of a sterile substrate prepared with hard-cooked cheeses, and identified the time of ripening when main changes in proteolysis are produced. The extract, a representative model of the aqueous phase of the cheeses, was obtained from Reggianito cheeses of different ripening times (3, 90, and 180 days) made with starters composed of the strains tested, either SF138 or SF209. To obtain the substrate, the cheese was extracted with water, then centrifuged and the aqueous phase was sterilized by filtration through membrane (0.45 ??m). The substrates were incubated at 34 °C during 21 days; samples were taken at 0, 3, 7, 14, and 21 days. Sterility was verified by plating samples on skim milk agar and incubating at 37 °C for 48 h. Proteolysis was determined by liquid chromatography of soluble peptides and free amino acids. Great variation in peptide profiles was found as incubation progressed in cheese extracts, which evidenced that proteases and peptidases from the starter were active and able to degrade the proteinaceous material available in the extracts. The extracts derived from cheeses with L. helveticus SF138 showed low production of peptides and a notable increase in free amino acids content during incubation. L. helveticus SF209, on the contrary, caused an increase on soluble peptides, but the free amino acids accumulation was lower than in the first case, which suggested that L. helveticus SF209 had either a low peptydolitic activity or produced an intense amino acids breakdown. This trend was more evident for extracts prepared with 90-day-old cheeses. It was concluded that the strains of L. helveticus assayed showed potentially complementary proteolytic abilities, as SF209 was able to provide a continuous replenishment of peptides during incubation, while SF138 increased their hydrolysis to free amino acids. The extract was an appropriate medium to model hard cooked cheese ripening in short periods of time.     

Influence of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus on Mozzarella cheese and cheese whey

We investigated the effect of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus starter bacteria on Mozzarella cheese functionality and whey viscosity. Mozzarella cheeses were manufactured with Lactobacillus helveticus LH100 paired with one of four S. thermophilus strains: MR-1C, a bacterium that produces a capsular exopolysaccharide; MTC360, a strain that secretes a ropy exopolysaccharide; TAO61, a nonexopolysaccharide-producing commercial cheese starter; and DM10, a nonencapsulated, exopolysaccharide-negative mutant of strain MR-1C. As expected, cheese moisture levels were significantly higher in Mozzarella cheeses made with exopolysaccharide-positive versus exopolysaccharide-negative streptococci, and melt properties were better in the higher moisture cheeses. Whey viscosity measurements showed that unconcentrated and ultrafiltered, fivefold concentrated whey from cheeses made with S. thermophilus MTC360 were significantly more viscous than whey from cheeses made with MR-1C, TAO61, or DM10. No significant differences were noted between the viscosity of unconcentrated or concentrated whey from cheeses made with S. thermophilus MR-1C versus the industrial cheese starter TAO61. These data indicate that encapsulated, but not ropy, exopolysaccharide-producing S. thermophilus strains can be utilized to increase the moisture level of cheese and to improve the melt properties of Mozzarella cheese without adversely affecting whey viscosity.     

Starter strain related effects on the biochemical and sensory properties of Cheddar cheese

A detailed investigation was undertaken to determine the effects of four single starter strains, Lactococcus lactis subsp. lactis 303, Lc. lactis subsp. cremoris HP, Lc. lactis subsp. cremoris AM2, and Lactobacillus helveticus DPC4571 on the proteolytic, lipolytic and sensory characteristics of Cheddar cheese. Cheeses produced using the highly autolytic starters 4571 and AM2 positively impacted on flavour development, whereas cheeses produced from the poorly autolytic starters 303 and HP developed off-flavours. Starter selection impacted significantly on the proteolytic and sensory characteristics of the resulting Cheddar cheeses. It appeared that the autolytic and/or lipolytic properties of starter strains also influenced lipolysis, however lipolysis appeared to be limited due to a possible lack of availability or access to suitable milk fat substrates over ripening. The impact of lipolysis on the sensory characteristics of Cheddar cheese was unclear, possibly due to minimal differences in the extent of lipolysis between the cheeses at the end of ripening. As anticipated seasonal milk supply influenced both proteolysis and lipolysis in Cheddar cheese. The contribution of non-starter lactic acid bacteria towards proteolysis and lipolysis over the first 8 months of Cheddar cheese ripening was negligible.     

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.     

Selection and use of autochthonous multiple strain cultures for the manufacture of high-moisture traditional Mozzarella cheese

Lactobacillus plantarum 18A, Lactobacillus helveticus 2B, Lactobacillus delbrueckii subsp. lactis 20F, Streptococcus thermophilus 22C, Enterococcus faecalis 32C and Enterococcus durans 16E were the most acidifying strains within 146 isolates for natural whey starters. The effect of media and temperature on 2 autochthonous multiple strain cultures (AMSI: 18A, 2B, 20F and 22C, 32C and 16E and AMSII: 18A, 2B, 20F and 22C) was studied. Genomic analysis showed a constant cell numbers for AMSII during 16 days of propagation in whey milk. Mozzarella cheese was made by using AMSII, commercial starter (CS) or citric acid (DA). Compared to other cheeses, the DA had a lower level of protein, ash, Ca, free amino acids and a higher level of moisture. Based on confocal laser scanning microscopy analysis, AMSII cheese showed the lowest microstructural variations during the period of storage compared to other cheeses. All the sensory attributes were scored highest for AMSII cheese. ASMII extend the shelf-life to ca. 12-15 days instead of the 5-7 days of traditional high-moisture Mozzarella cheese.     

Growth,survival, and peptidolytic activity of Lactobacillus plantarum I91 in a hard-cheese model

In this work, we studied the growth, survival, and peptidolytic activity of Lactobacillus plantarum I91 in a hard-cheese model consisting of a sterile extract of Reggianito cheese. To assess the influence of the primary starter and initial proteolysis level on these parameters, we prepared the extracts with cheeses that were produced using 2 different starter strains of Lactobacillus helveticus 138 or 209 (Lh138 or Lh209) at 3 ripening times: 3, 90, and 180 d. The experimental extracts were inoculated with Lb. plantarum I91; the control extracts were not inoculated and the blank extracts were heat-treated to inactivate enzymes and were not inoculated. All extracts were incubated at 34°C for 21 d, and then the pH, microbiological counts, and proteolysis profiles were determined. The basal proteolysis profiles in the extracts of young cheeses made with either strain tested were similar, but many differences between the proteolysis profiles of the extracts of the Lh138 and Lh209 cheeses were found when riper cheeses were used. The pH values in the blank and control extracts did not change, and no microbial growth was detected. In contrast, the pH value in experimental extracts decreased, and this decrease was more pronounced in extracts obtained from either of the young cheeses and from the Lh209 cheese at any stage of ripening. Lactobacillus plantarum I91 grew up to 8 log during the first days of incubation in all of the extracts, but then the number of viable cells decreased, the extent of which depended on the starter strain and the age of the cheese used for the extract. The decrease in the counts of Lb. plantarum I91 was observed mainly in the extracts in which the pH had diminished the most. In addition, the extracts that best supported the viability of Lb. plantarum I91 during incubation had the highest free amino acids content. The effect of Lb. plantarum I91 on the proteolysis profile of the extracts was marginal. Significant changes in the content of free amino acids suggested that the catabolism of free amino acids by Lb. plantarum I91 prevailed in a weakly proteolyzed medium, whereas the release of amino acids due to peptidolysis overcame their catabolism in a medium with high levels of free amino acids. Lactobacillus plantarum I91 was able to use energy sources other than lactose to support its growth because equivalent numbers of cells were observed in extracts containing residual amounts of lactose and in lactose-depleted extracts. The contribution of Lb. plantarum I91 to hard-cooked cheese peptidolysis was negligible compared with that of the starter strain; however, its ability to transform amino acids is a promising feature of this strain.     

The effect of using an exopolysaccharide‐producing culture on the physicochemical properties of low‐fat and reduced‐fat Kasar cheeses

A mixed starter culture containing exopolysaccharide (EPS)‐producing strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus was combined with Lactobacillus helveticus LH301 and used in the manufacture of low‐fat and reduced‐fat Kasar cheeses. For comparison, low‐fat (C10) and reduced‐fat (C20) cheeses were made using EPS‐producing (EPS⁺) starter strain and EPS‐non‐producing (EPS^?) starter strain. The physicochemical properties of the cheeses were assessed in terms of chemical composition, texture, microstructure and microbial content over 90 days. Cheeses made with EPS‐producing culture (EPS10 and EPS20) had lower protein contents than control cheeses with 10% and 20% fat in dry basis (C10 and C20). Scanning electron microscopy images showed that using EPS‐producing culture resulted in a less compact protein matrix and sponge‐like structure in the cheese samples. In general, cheeses made using EPS‐producing culture had lower total viable counts. This could be related to the reduced survivability of EPS‐producing cells in the cheese matrix during ripening due to autolysis ability.     

Ripening of cheddar cheese with added attenuated adjunct cultures of lactobacilli

We made Milled curd Cheddar cheese with Lactococcus starter and an adjunct culture of Lactobacillus helveticus I or Lactobacillus casei T subjected to different attenuation treatments: freeze shocking (FS), heat shocking (HS), or spray drying (SD). Proteolysis during cheese ripening (0 to 6 mo), measured by urea-PAGE and water-soluble nitrogen, indicated only minor differences between control and most adjunct-treated cheeses. However, there were significant differences in the effect of Lactobacillus adjuncts on the level of free amino nitrogen in cheese. Cheeses made with FS or HS Lb. helveticus adjunct exhibited significantly greatest rates of free amino group formation. Lipolysis as measured by total free fatty acids was consistently highest in adjunct-treated cheeses, and FS Lb. casei-treated cheeses showed the highest rate of free fatty acid formation followed by FS Lb. helveticus treated cheeses. Mean flavor and aroma scores were significantly higher for cheeses made with Lb. helveticus strain. Freeze-shocked Lb. helveticus-treated cheeses obtained the highest flavor and aroma scores. Sensory evaluation indicated that most of the adjunct-treated cheeses promoted better texture and body quality.     

Biochemical patterns in ovine cheese: Influence of probiotic strains

This study was undertaken to evaluate the effect of lamb rennet paste containing probiotic strains on proteolysis, lipolysis, and glycolysis of ovine cheese manufactured with starter cultures. Cheeses included control cheese made with rennet paste, cheese made with rennet paste containing Lactobacillus acidophilus culture (LA-5), and cheese made with rennet paste containing a mix of Bifidobacterium lactis (BB-12) and Bifidobacterium longum (BB-46). Cheeses were sampled at 1, 7, 15, and 30 d of ripening. Starter cultures coupled with probiotics strains contained in rennet paste affected the acidification and coagulation phases leading to the lowest pH in curd and cheese containing probiotics during ripening. As consequence, maturing cheese profiles were different among cheese treatments. Cheeses produced using rennet paste containing probiotics displayed higher percentages of α_S1-I-casein fraction than traditional cheese up to 15 d of ripening. This result could be an outcome of the greater hydrolysis of α-casein fraction, attributed to higher activity of the residual chymosin. Further evidence for this trend is available in chromatograms of water-soluble nitrogen fractions, which indicated a more complex profile in cheeses made using lamb paste containing probiotics versus traditional cheese. Differences can be observed for the peaks eluted in the highly hydrophobic zone being higher in cheeses containing probiotics. The proteolytic activity of probiotic bacteria led to increased accumulation of free amino acids. Their concentrations in cheese made with rennet paste containing Lb. acidophilus culture and cheese made with rennet paste containing a mix of B. lactis and B. longum were approximately 2.5 and 3.0 times higher, respectively, than in traditional cheese. Principal component analysis showed a more intense lipolysis in terms of both free fatty acids and conjugated linoleic acid content in probiotic cheeses; in particular, the lipolytic pattern of cheeses containing Lb. acidophilus is distinguished from the other cheeses on the basis of highest content of health-promoting molecules. The metabolic activity of the cheese microflora was also monitored by measuring acetic, lactic, and citric acids during cheese ripening. Cheese acceptability was expressed for color, smell, taste, and texture perceived during cheese consumption. Use of probiotics in trial cheeses did not adversely affect preference or acceptability; in fact, panelists scored probiotic cheeses higher in preference over traditional cheese, albeit not significantly.     

Proteolysis texture and microstructure of low‐fat Tulum cheese affected by exopolysaccharide‐producing cultures during ripening

The objective of the study was to determine the effects of exopolysaccharide (EPS)‐producing or non‐EPS‐producing starters on proteolysis, physical and microstructural characteristics of full‐fat or low‐fat Tulum cheeses during ripening. For this purpose, Tulum cheese was manufactured using full‐ or low‐fat milk with EPS‐producing and non‐EPS‐producing starter cultures. Chemical composition, proteolysis, texture profiles and microstructure of the cheeses were studied during 90 days of ripening. Urea‐PAGE of water‐insoluble and RP‐HPLC peptide profiles of water‐soluble fractions of the cheeses showed that the use of starters resulted in different degradation patterns in all cheeses during ripening. Although β‐casein exhibited similar degradation patterns in all cheeses, small differences are present in α_s1‐casein degradation during ripening. Reducing fat in Tulum cheese changed the RP‐HPLC peptide profile of the cheeses. The use of EPS‐producing cultures improved the textural characteristics and changed the microstructure and proteolysis of low‐fat Tulum cheese.     

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.     

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.     

Multivariate analysis of proteolysis patterns differentiated the impact of six strains of probiotic bacteria on a semi-hard cheese

The individual contribution of 6 strains of probiotic bacteria (3 of Lactobacillus acidophilus and 3 of the Lactobacillus casei group) to proteolysis patterns in a semi-hard cheese was assessed. Control cheeses (without probiotics) and 2 types of experimental cheeses (with the addition of probiotics either directly to milk or by a 2-step fermentation method) were manufactured. Cheeses containing Lb. acidophilus showed the most extensive peptidolysis, which was evidenced by changes in the peptide profiles and a noticeable increase of free amino acids compared with control cheeses. The strains of the Lb. casei group showed a lower contribution to cheese peptidolysis, which consisted mainly of free amino acid increase. Two-step fermentation improved peptidolytic activity for only one of the cultures of Lb. acidophilus tested. The addition of Lb. acidophilus strains into cheese may be suitable not only for their beneficial health effect but also for their influence on secondary proteolysis, consistent with acceleration of ripening and improved flavor formation.     

Influence of a defined-strain starter and Lactobacillus plantarum as adjunct culture on volatile compounds and sensory characteristics of Manchego cheese

Three batches of Manchego cheese were manufactured using one of the following starter culture systems: (1) a defined strain starter culture comprising Lactococcus lactis subsp. lactis and Leuconostoc mesenteroides subsp. dextranicum; (2) the above-defined strain starter culture and an adjunct culture (Lactobacillus plantarum), all these strains being isolated from high-quality Manchego cheeses and (3) a commercial starter consisting of two strains of Lactococcus lactis. Differences in volatile profile and the sensory characteristics of these cheeses were studied. After 4 months of ripening, the two batches of cheese made with the defined strain starter cultures obtained the highest scores for sensory attributes and for the overall impression. Additionally, Purge & Trap and SDE analysis showed a more complex volatile profile in these cheeses than in those made with the commercial starter. Extending the maturation time to 8 months for cheeses made with the defined starter cultures led to significant higher levels of free fatty acids and ethyl esters in those cheeses made without adjunct culture. However, panelists did not find significant differences among the sensory characteristics of the two cheeses.     

Preliminary observations on proteolysis in Manchego cheese made with a defined-strain starter culture and adjunct starter (Lactobacillus plantarum) or a commercial starter

Different authors have demonstrated the potential of adding lactobacilli as adjunct cultures to pasteurized milk used in cheese manufacture. The aim of this work was to observe the effect of the use of a defined-strain starter culture and the addition of an adjunct culture (Lactobacillus plantarum) to cheesemilk in order to determine their effect on the ripening of Manchego cheese. Manchego cheeses were manufactured using one of the following starter culture systems: a defined starter consisting of Lactococcus lactis ssp. lactis and Leuconostoc mesenteroides ssp. dextranicum; a defined starter, as described above, and Lb. plantarum, which were isolated from a good quality Manchego cheese made from raw milk, or a commercial starter comprised of two strains of Lc. lactis. The cheeses were sampled at 15, 45, 90 and 150 d of ripening. Principal component analysis of peak heights of reversed-phase HPLC chromatograms of 70% (v/v) ethanol-insoluble and -soluble fractions distributed the samples according to the starter used in their manufacture. Quantitative differences in several peptides were evident between the three cheeses. Cheeses made with the defined-strain starters received higher scores for the flavour quality and intensity and for overall impression than the cheeses made with the commercial starter.     

Influence of microfiltration and adjunct culture on quality of Domiati cheese

The effects of microfiltration and pasteurization processes on proteolysis, lipolysis, and flavor development in Domiati cheese during 2 mo of pickling were studied. Cultures of starter lactic acid bacteria isolated from Egyptian dairy products were evaluated in experimental Domiati cheese for flavor development capabilities. In the first trial, raw skim milk was microfiltered and then the protein:fat ratio was standardized using pasteurized cream. Pasteurized milk with same protein:fat ratio was also used in the second trial. The chemical composition of cheeses seemed to be affected by milk treatment—microfiltration or pasteurization—rather than by the culture types. The moisture content was higher and the pH was lower in pasteurized milk cheeses than in microfiltered milk cheeses at d 1 of manufacture. Chemical composition of experimental cheeses was within the legal limits for Domiati cheese in Egypt. Proteolysis and lipolysis during cheese pickling were lower in microfiltered milk cheeses compared with pasteurized milk cheeses. Highly significant variations in free amino acids, free fatty acids, and sensory evaluation were found among the cultures used in Domiati cheesemaking. The cheese made using adjunct culture containing Lactobacillus delbrueckii ssp. lactis, Lactobacillus paracasei ssp. paracasei, Lactobacillus casei, Lactobacillus plantarum, and Enterococcus faecium received high scores in flavor acceptability. Cheeses made from microfiltered milk received a higher score in body and texture compared with cheeses made from pasteurized milk.     

Comparison of Fascal cheese produced with natural,commercial or autochthonous cultures

Lactic acid bacteria were isolated from raw ovine milk and Fascal cheese and used as two alternative inoculants for production of this Brazilian cheese. Microbiological counts and moisture of these cheeses were comparable to those observed in cheese made with a commercial starter. All cheeses showed absence of Salmonella spp., tolerable counts of coliforms, but coagulase‐positive staphylococci counts exceeded the standard values. Cheeses produced with Lactobacillus plantarum LCN 28 and Lactobacillus rhamnosus LCN 43 showed better results than those observed for the other experimental combinations. Autochthonous cultures could be beneficial to the manufacture of high quality typical raw milk cheese.