Characterization of yeasts involved in the ripening of Pecorino Crotonese cheese

The aims of this work were to identify and characterize for some important technological properties the yeast species present throughout the ripening process of Pecorino Crotonese, a traditional cheese produced in a well defined area of Southern Italy. In particular, the strain technological properties considered include fermentation/assimilation of galactose and lactose, assimilation of lactate and citrate in the presence of different NaCl concentrations, hydrolysis of butter fat, skim milk, gelatine and casein, production of brown pigments in cheese agar and ability to produce biogenic amines. High yeast levels were recorded in cheese samples already after 5 h of brining (about 5 log cfu/g) and these concentration remained constant during ripening. The yeast isolates belonged to restrict number of yeast species. While Kluyveromyces lactis and Saccharomyces cerevisiae were isolated prevalently in the first stages of Pecorino Crotonese production, Yarrowia lipolytica and Debaryomyces hansenii dominated during the later stages of maturation. Otherwise, the latter two were very NaCl resistant species. In fact, D. hansenii strains conserved the ability to assimilate lactose and galactose in the presence of 10% NaCl, while almost all the strains of Y. lipolytica isolated assimilated citrate and lactate up to 7.5% NaCl. Y. lipolytica isolates evidenced also the highest proteolytic and lipolytic activities and the capability to catabolize tyrosine producing brown pigment. In addition they resulted in the highest aminobiogenic potential decarboxylating ornithine, phenylalanine, tyrosine and lysine. However, they were not able to produce histamine, biogenic amine produced by three strains of D. hansenii.     

Taxonomical and technological characteristics of Saccharomyces spp. associated with blue veined cheese

In blue veined cheeses, the dominant yeast species in most cases is Debaryomyces hansenii. Saccharomyces spp. occurs less frequently, but they can be found in some blue veined cheeses. In the present study, the taxonomy of Saccharomyces spp. associated to blue veined cheeses was studied and comparisons made to type strains of Saccharomyces spp. and starter cultures of Saccharomyces spp. used in other food fermentations. Phenotypically, the cheese strains were referred to the Saccharomyces sensu stricto complex and were further identified as S. cerevisiae. Genotypically, the Saccharomyces spp. investigated were similar although chromosomal polymorphism were observed. Concerning the technological characteristics, they were similar in assimilation and fermentation of the residual sugars and organic acids naturally found in cheese. The investigated yeasts were also similar in their lipolytic activity being able to hydrolyse tributyrin and low chain (C:8), but not C:14 fatty acids. However, they differed in their tolerance to NaCl with the blue cheese strains showing a higher tolerance. The cheese strain S. cerevisiae FB 7 was the only yeast capable of degrading casein. It mainly degraded the alpha(s1)-casein and the beta(alpha2)-casein components. It was also the only isolate stimulating the development of Penicillium roqueforti in cheese agar imitating the conditions in blue veined cheese. The stimulation of P. roqueforti was most pronounced for the least proteolytic strain of P. roqueforti examined.     

Saccharomyces cerevisiae as a starter culture in Mycella

The potential use of Saccharomyces cerevisiae FB7 as an additional starter culture for the production of Mycella, a Danish Gorgonzola type cheese, was investigated. Two dairy productions of Mycella, each containing batches of experimental cheeses with S. cerevisiae added and reference cheeses without yeast added were carried out. For both experimental and reference cheeses, chemical analysis (pH, a(w), NaCl, water and fat content) were carried out during the ripening period, but no significant differences were found. The evolution of lactic acid bacteria was almost identical in both the experimental and reference cheeses and similar results were found for the number of yeast. S. cerevisiae FB7 was found to be predominant in the core of the experimental cheeses throughout the ripening period, while Debaryomyces hansenii dominated in the reference cheese and on the surface of the experimental cheeses. In the cheeses with S. cerevisiae FB7, an earlier sporulation and an improved growth of Penicillium roqueforti was observed compared to the reference cheeses. Furthermore, in the experimental cheese, synergistic interactions were also found in the aroma analysis, the degradation of casein and by the sensory analysis. The observed differences indicate a positive contribution to the overall quality of Mycella by S. cerevisiae FB7.     

Cheese yeasts

Numerous traditionally aged cheeses are surface ripened and develop a biofilm, known as the cheese rind, on their surfaces. The rind of such cheeses comprises a complex community of bacterial and fungal species that are jointly responsible for the typical characteristics of the various cheese varieties. Surface ripening starts directly after brining with the rapid colonization of the cheese surface by yeasts. The initially dominant yeasts are acid and salt-tolerant and are capable of metabolizing the lactate produced by the starter lactic acid bacteria and of producing NH₃ from amino acids. Both processes cause the pH of the cheese surface to rise dramatically. This so-called deacidification process enables the establishment of a salt-tolerant, Gram-positive bacterial community that is less acid-tolerant. Over the past decade, knowledge of yeast diversity in cheeses has increased considerably. The yeast species with the highest prevalence on surface-ripened cheeses are Debaryomyces hansenii and Geotrichum candidum, but up to 30 species can be found. In the cheese core, only lactose-fermenting yeasts, such as Kluyveromyces marxianus, are expected to grow. Yeasts are recognized as having an indispensable impact on the development of cheese flavour and texture because of their deacidifying, proteolytic, and/or lipolytic activity. Yeasts are used not only in the production of surface-ripened cheeses but also as adjunct cultures in the vat milk in order to modify ripening behaviour and flavour of the cheese. However, yeasts may also be responsible for spoilage of cheese, causing early blowing, off-flavour, brown discolouration, and other visible alterations of cheese.     

Control of Listeria monocytogenes in raw-milk cheeses

The development of Listeria monocytogenes in cheeses made with raw-milk originating from six different farms and according to the Saint-Nectaire cheesemaking technology was studied. Milk was inoculated with two strains of L. monocytogenes at 5 to 10 CFU/25 ml. Microbial and chemical analyses were carried out at appropriate intervals during ripening. L. monocytogenes did not grow in the cores of cheeses prepared with milk originating from three farms. That inhibition could be partially attributed to the pH values and L-lactate content. There was no growth in cheeses with pH below 5.2 and lactate content around 14 mg/g. In all cheeses, L. monocytogenes stopped growing in the cores of cheeses after eight days and some other factors may be involved in the inhibition. No relation was found between L. monocytogenes count and other microbial counts. Growth occurred on cheese surfaces between eight and eighteen days, when the pH significantly increased. The lowest L. monocytogenes growth was found on the surface of cheeses with the lowest pH and without any core growth. Further studies will be performed to clarify the involvement of the microbial community in L. monocytogenes inhibition, in particular during the ripening period.     

Behavior of Brevibacterium linens and Debaryomyces hansenii as ripening flora in controlled production of smear soft cheese from reconstituted milk: growth and substrate consumption dairy foods

Experimental cheeses inoculated with Debaryomyces hansenii and Brevibacterium linens were ripened for 76 d under aseptic conditions. Triplicate cheese-making trials were similar as a result of efficient control of the atmosphere. In all trials, D. hansenii grew rapidly during the first 2 d and then slowed, but growth remained exponential until d 10 (generation time around 70 h). Total cell counts were higher than the number of viable cells, and after 10 d they remained around 3 x 10(9) yeast/g of DM. This difference resulted from the nonviability of a fraction of D. hansenii. After d 15, the pH of the rind was close to 7, and B. linens grew exponentially until d 25 (generation time around 70 h). The growth rate subsequently decreased but remained exponential (generation time around 21 d). Cell counts of D. hansenii and B. linens were correlated with the environmental technical conditions. Total D. hansenii counts were also correlated with total B. linens counts. Viable B. linens counts were related to rind lactate, and total counts depended on rind pH, internal lactate, and D. hansenii viable counts. The internal pH of the cheese depended on lactate concentrations, whereas surface pH was related to internal lactose, temperature, and relative humidity. These results suggest a determining role of the diffusion of the carbon sources in the ripening of smear soft cheese.     

DNA typing methods for differentiation of Debaryomyces hansenii strains and other yeasts related to surface ripened cheeses

The discriminative power of ITS-PCR, ITS-PCR RFLP and mitochondrial (mt)-DNA RFLP were evaluated for differentiation of yeasts of importance for surface ripened cheeses. In total 60 isolates were included. Of these, 40 strains of the following species, Debaryomyces hansenii var. hansenii, D. hansenii var. fabryi, Saccharomyces cerevisiae, Candida zeylanoides, Kluyveromyces lactis and Yarrowia lipolytica, were obtained from culture collections and 20 isolates of D. hansenii representing six different phenotypes were collected from seven Danish producers of surface ripened cheeses. ITS-PCR was evaluated for differentiation at species level on the 40 strains obtained from culture collections. Ten strains of each variety of D. hansenii and five strains of each of the above mentioned species were analysed. For each of the investigated species, a specific ITS1-5.8S rDNA-ITS2 region size was observed. Accordingly ITS-PCR was found valuable for differentiation at species level of yeasts of importance for surface ripened cheeses. ITS-PCR RFLP was investigated for the purpose of strain typing of D. hansenii. Ten CBS strains of each variety of D. hansenii were analysed. Only one enzyme (TaqI) out of several investigated (BamHI, DpnI, Fnu4HI, HaeIII, HindIII, HpaII, NlaII, Sau3AI, TaqI) demonstrated genetic diversity within the strains. This enzyme divided the 20 strains in three groups. Sequence analysis of the ITS1-5.8S rDNA-ITS2 region for the type strains of each variety of D. hansenii showed an identity of 99.84%, corresponding to a difference in one basepair. Based on these results, ITS-PCR RFLP was found ineffective for strain typing of D. hansenii. MtDNA RFLP using HaeIII and HpaII was evaluated for strain typing of D. hansenii on the 20 CBS strains of D. hansenii. The CBS strains were divided into 16 groups according to their restriction profiles, which proved the method useful for typing of D. hansenii at subspecies level. The 20 dairy isolates showed a lower genetic variability than the CBS strains as they were divided into eight groups. Cluster analysis of the 20 CBS strains and the 20 dairy isolates based on their mtDNA restriction profiles showed (max. similarity level = 52%) that the dairy isolates only clustered with the CBS strains of D. hansenii var. hansenii. For some of the dairies more than one strain of D. hansenii were found to be involved in the ripening process, indicating that the method could be useful for subspecies typing and investigation of the microbial succession between strains of D. hansenii during the ripening process of surface ripened cheeses.     

Occurrence and characterization of yeasts isolated from artisanal Fiore Sardo cheese

The occurrence of yeast microflora in artisanal Fiore Sardo cheese during ripening was studied. Mean yeast counts ranged from 2.64+/-1 log(10) cfu ml(-1) in milk to 0.65+/-1 log(10) cfu g(-1) in 9 months cheese, with the higher counts observed in 48-h-old cheese. Strains belonging to the prevalent species Debaryomyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Candida zeylanoides and Candida lambica were selected for technological and genotypic characterization. All D. hansenii strains fermented glucose and assimilated lactate, a high percentage assimilated citrate and only a few showed proteolytic and lipolytic activity. All K. lactis strains were able to both assimilate and ferment lactose, to assimilate lactate and to exhibit proteolytic activity on casein. G. candidum assimilated lactate and some strains showed proteolytic and lipolytic activity. C. zeylanoides showed lipolytic activity on tweens and the majority of strains assimilated citrate. C. lambica fermented glucose and assimilated lactate. Considering their diffusion and technological characteristics, an important role for K. lactis and G. candidum in the early stages of the ripening process and for D. hansenii after the first month of ripening can be suggested. RAPD-PCR analysis with M13 primer grouped the isolates in well-separated clusters with their type strains and confirmed the previous phenotypic identification. The high intraspecific homogeneity observed in tested strains could be explained by their isolation from a common substrate and from neighbouring geographical areas. This preliminary study allowed us to isolate autochthon yeast strains showing particular properties which can contribute to the production of typical cheese taste and flavour.     

Ammonia production and its possible role as a mediator of communication for Debaryomyces hansenii and other cheese-relevant yeast species

Ammonia production by yeasts may contribute to an increase in pH during the ripening of surface-ripened cheeses. The increase in pH has a stimulatory effect on the growth of secondary bacterial flora. Ammonia production of single colonies of Debaryomyces hansenii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Geotrichum candidum was determined on glycerol medium (GM) agar and cheese agar. The ammonia production was found to vary, especially among yeast species, but also within strains of D. hansenii. In addition, variations in ammonia production were found between GM agar and cheese agar. Ammonia production was positively correlated to pH measured around colonies, which suggests ammonia production as an additional technological parameter for selection of secondary starter cultures for cheese ripening. Furthermore, ammonia appeared to act as a signaling molecule in D. hansenii as reported for other yeasts. On GM agar and cheese agar, D. hansenii showed ammonia production oriented toward neighboring colonies when colonies were grown close to other colonies of the same species; however, the time to oriented ammonia production differed among strains and media. In addition, an increase of ammonia production was determined for double colonies compared with single colonies of D. hansenii on GM agar. In general, similar levels of ammonia production were determined for both single and double colonies of D. hansenii on cheese agar.     

Aspects of enzymology and biochemical properties of Brevibacterium linens relevant to cheese ripening: a review.

Brevibacterium linens is a major surface microorganism that is present in the smear of surface-ripened cheeses. The enzymology and biochemical characteristics of B. linens influence the ripening and final characteristics of smear surface-ripened cheeses. Proteolytic, peptidolytic, esterolytic, and lipolytic activities, which are of particular importance in the ripening process, are discussed in detail. This review also describes the production of volatile compounds, especially sulfur-containing ones, by B. linens, which are thought to be important in respect to the flavor of smear surface-ripened cheeses. The unique orange-colored carotenoids and the factors effecting their production by B. linens are also presented. The catabolism of aromatic amino acids, bacteriocin production, plasmids, and miscellaneous biochemical and physiological properties (peptidoglycan type, antibiotic resistance, insecticide degradation, and biotechnological applications) of B. linens are discussed. The problem associated with the current taxonomical classification of B. linens strains caused by strain variation is evaluated. Finally, the application of B. linens cell extracts or its proteolytic enzymes as cheese ripening accelerants for semi-hard or hard cheese varieties is considered.     

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.     

Properties and antimicrobial activity of the smear surface cheese coryneform bacterium <Emphasis Type="Italic">Brevibacterium linens</Emphasis>

Surface microorganisms contribute to the ripening of some low-moisture cheese varieties and the composition of the surface microflora is dynamic. Brevibacterium linens is an important surface microorganism that is present in the smear of surface-ripened cheeses and is commonly regarded as the organism primarily responsible for the characteristic taste, aroma, and color of surface cheese. The enzymology and biochemical characteristics of B. linens influence the ripening and final characteristics of smear surface-ripened cheeses. Proteolytic, peptidolytic, esterolytic, and lipolytic activities are of particular importance in the ripening process. Because of its putative importance to the ripening in smear-ripened cheeses, B. linens is the best studied component of the microflora, although in comparision with other dairy-related microorganisms, it is poorly characterized. B. linens produces antimicrobial substances that inhibit the growth of many food poisoning bacteria as well as several yeast and moulds. Some inhibitory substances produced by this species were identified as bacteriocins. Bacteriocins could appear as potential agents to be applied in food conservation systems in order to provide microbiologically stable foods. This article describes the properties of B. linens and discusses about the potential of this species to produce bacteriocins and other antimicrobial substances, which are important for production of high quality cheese.     

Yeasts as adjunct starters in matured Cheddar cheese

Debaryomyces hansenii and Yarrowia lipolytica are typical foodborne yeast species frequently associated with dairy products and capable of predominating the yeast composition in such systems. The two species fulfil a number of criteria to be regarded as co-starters for cheesemaking. They are known for their proteolytic and lipolytic activity as well as their compatibility and stimulating action with the lactic acid starter cultures when co-inoculated. Recent studies indicated that yeasts could be included as part of starter cultures for the manufacturing of cheese, enhancing flavour development during the maturation. The potential of D. hansenii and Y. lipolytica as agents for accelerated ripening of matured Cheddar cheese has been evaluated during four cheese treatments. The interaction between the two yeast species and the lactic acid bacteria was surveyed incorporating (i) D. hansenii, (ii) Y. lipolytica, (iii) both species as adjuncts to the starter culture and (iv) a control cheese without any additions for the production of matured Cheddar cheese. The physical and chemical properties of the cheeses were monitored in order to evaluate the contribution of the yeasts to cheese maturation. The yeasts grew in association with the lactic acid bacteria without any inhibition. The yeasts species when individually added contributed to the development of bitter flavours despite accelerated development of strong Cheddar flavours. When both species were incorporated as part of the starter culture, the cheese, however, had a good strong flavour after a reduced ripening period. The cheese retained this good flavour and aroma after 9 months of production. The simultaneous application of D. hansenii and Y. lipolytica as part of the starter culture for the production of matured Cheddar cheese is proposed.     

Microbial, chemical and sensorial variables of the Spanish traditional blue-veined Cabrales cheese, as affected by inoculation with commercial Penicillium roqueforti spores

This paper reports the technological effects of inoculating Cabrales cheese (a traditional, Spanish, blue-veined cheese) with Penicillium roqueforti spores. Three batches of inoculated Cabrales cheese were manufactured and a number of their microbial and biochemical variables recorded. The results were compared with those obtained for three batches of control cheese made using traditional technology (i.e., adding neither starter cultures nor fungal spores). Although mould and yeast populations grew more quickly in the inoculated cheeses, their normally dominant and representative microbial populations were not affected neither was their gross biochemical composition changed. The variations observed were thought to be caused by the uncontrolled environmental conditions of manufacture and ripening. The development of free amino acids and volatile compounds was significantly increased at 30 days in the inoculated cheeses, although the values for both types of cheese were almost identical at 90 days. The inoculated cheeses obtained higher scores in a hedonistic sensorial evaluation. Thus, inoculation improved the standardization and quality of the cheese.     

Yeasts associated with Sardinian ewe's dairy products

In the present work, the occurrence of yeasts in different types of typical Sardinian ewe's cheeses (32 samples of pecorino, 32 of caciotta, 40 of feta, 56 of ricotta) was determined. For the strains isolated the following properties were studied: proteolytic and lipolytic activities, the ability to grow at different temperatures, different concentrations of salt, and to assimilate and/or ferment compounds like lactate, citrate, lactose, glucose, galactose, lactic acid. Of 160 samples analysed, 76.2% yielded growth of yeasts. Yeast counts showed a certain variability among the samples. The highest levels were observed in caciotta and feta cheeses. A total of 281 strains belonging to 16 genera and 25 species were identified. In general, Debaryomyces hansenii was the dominant species, representing 28.8% of the total isolates. Other frequently appearing species were Geotrichum candidum, Kluyveromyces lactis and K. marxianus. Other genera encountered were Pichia, Candida, Dekkera, Yarrowia and Rhodotorula. With regard to the biochemical and technological properties of the yeasts, only K. lactis, K. marxianus and Dek. anomala assimilated and fermented lactose, whereas the majority of the species assimilated lactic acid. The assimilation of citrate was a characteristic of D. hansenii, R. rubra and Y. lipolytica. On the whole, the yeasts were weakly proteolytic while lipolytic activity was present in several species. A high percentage of strains showed a certain tolerance to low temperatures while only some strains of D. hansenii and K. lactis were able to grow at a 10% NaCl concentration.     

Influence of starters on chemical, biochemical, and sensory changes in Turkish White-brined cheese during ripening

Turkish White-brined cheese was manufactured using Lactococcus strains (Lactococcus lactis ssp. lactis NCDO763 plus L. lactis ssp. cremoris SK11 and L. lactis ssp. lactis UC317 plus L. lactis ssp. cremoris HP) or without a starter culture, and ripened for 90 d. It was found that the use of starters significantly influenced the physical, chemical, biochemical, and sensory properties of the cheeses. Chemical composition, pH, and sensory properties of cheeses made with starter were not affected by the different starter bacteria. The levels of soluble nitrogen fractions and urea-PAGE of the pH 4.6-insoluble fractions were found to be significantly different at various stages of ripening. Urea-PAGE patterns of the pH 4.6-insoluble fractions of the cheeses showed that considerable degradation of α_s1-casein occurred and that β-casein was more resistant to hydrolysis. The use of a starter culture significantly influenced the levels of 12% trichloroacetic acid-soluble nitrogen, 5% phosphotungstic acid-soluble nitrogen, free amino acids, total free fatty acids, and the peptide profiles (reverse phase-HPLC) of 70% (vol/vol) ethanol-soluble and insoluble fractions of the pH 4.6-soluble fraction of the cheeses. The levels of peptides in the cheeses increased during the ripening period. Principal component and hierarchical cluster analyses of electrophoretic and chromatographic results indicated that the cheeses were significantly different in terms of their peptide profiles and they were grouped based on the use and type of starter and stage of ripening. Levels of free amino acid in the cheeses differed; Leu, Glu, Phe, Lys, and Val were the most abundant amino acids. Nitrogen fractions, total free amino acids, total free fatty acids, and the levels of peptides resolved by reverse phase-HPLC increased during ripening. No significant differences were found between the sensory properties of cheeses made using a starter, but the cheese made without starter received lower scores than the cheeses made using a starter. It was found that the cheese made with strains NCDO763 plus SK11 had the best quality during ripening. It was concluded that the use of different starter bacteria caused significant differences in the quality of the cheese, and that each starter culture contributed to proteolysis to a different degree.     

Characterization of autochthonal yeasts isolated from Spanish soft raw ewe milk protected designation of origin cheeses for technological application

The yeasts involved in the ripening process of artisanal soft raw ewe milk Protected Designation of Origin (PDO) Torta del Casar and Queso de la Serena cheeses produced in Extremadura, Spain, were isolated throughout their ripening process, strain typed, and characterized for some important technological properties. A total of 508 yeast isolates were obtained and identified by inter-single sequence repeat anchored PCR amplification analysis and subsequent sequencing of the internal transcribed spacer ITS1/ITS2 5.8S rRNA. A total of 19 yeast species representing 8 genera were identified. Debaryomyces hansenii, Pichia kudriavzevii, Kluyveromyces lactis, and Yarrowia lipolytica were the predominant species. We selected 157 isolates, by genotyping and origin, for technological characterization. The evaluation of yeast isolates' growth under stress conditions of cheese ripening showed that 87 presented better performance. Among them, 71 isolates were not able to catabolize tyrosine to produce a brown pigment. Principal component analysis of the biochemical features of these isolates showed that 9 strains stood out, 3 K. lactis strains (2287, 2725, and 1507), 2 Pichia jadinii (1731 and 433), 2 Yarrowia alimentaria (1204 and 2150), Y. lipolytica 2495 and P. kudriavzevii 373. These strains displayed strong extracellular proteolytic activity on skim milk agar as well as an adequate enzymatic profile (strong aminopeptidase and weak protease activity), suggesting their great potential for cheese proteolysis. Extracellular lipolytic activity was mainly restricted to Yarrowia spp. isolates and weakly present in P. kudriavzevii 373 and K. lactis 2725, although enzymatic characterization by API-ZYM (bioMérieux SA) evidenced that all may contribute, at least in part, to the lipolysis process. Moreover, these strains were able to assimilate lactose, galactose, and glucose at NaCl concentrations higher than that usually found in cheese. However, lactate and citrate assimilation were limited to Y. lipolytica 2495, P. kudriavzevii 373, and P. jadinii 433, and may contribute to the alkalinizing process relevant to biochemical processes that take place in the last stages of ripening. By contrast, K. lactis strains showed acidifying capacity and β-galactosidase activity and may take part in the initial stages of ripening, together with lactic acid bacteria. Thus, considering the technological characteristics studied, the 9 selected strains presented biochemical features well suited to their potential use as adjunct cultures, alone or in combination with autochthonous starter bacteria in the cheesemaking process, to overcome the heterogeneity of these PDO cheeses, preserving their unique sensory characteristics.     

Nonstarter Lactobacillus strains as adjunct cultures for cheese making: in vitro characterization and performance in two model cheeses

Nonstarter lactic acid bacteria are the main uncontrolled factor in today's industrial cheese making and may be the cause of quality inconsistencies and defects in cheeses. In this context, adjunct cultures of selected lactobacilli from nonstarter lactic acid bacteria origin appear as the best alternative to indirectly control cheese biota. The objective of the present work was to study the technological properties of Lactobacillus strains isolated from cheese by in vitro and in situ assays. Milk acidification kinetics and proteolytic and acidifying activities were assessed, and peptide mapping of trichloroacetic acid 8% soluble fraction of milk cultures was performed by liquid chromatography. In addition, the tolerance to salts (NaCl and KCl) and the phage-resistance were investigated. Four strains were selected for testing as adjunct cultures in cheese making experiments at pilot plant scale. In in vitro assays, most strains acidified milk slowly and showed weak to moderate proteolytic activity. Fast strains decreased milk pH to 4.5 in 8 h, and continued acidification to 3.5 in 12 h or more. This group consisted mostly of Lactobacillus plantarum and Lactobacillus rhamnosus strains. Approximately one-third of the slow strains, which comprised mainly Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus curvatus, were capable to grow when milk was supplemented with glucose and casein hydrolysate. Peptide maps were similar to those of lactic acid bacteria considered to have a moderate proteolytic activity. Most strains showed salt tolerance and resistance to specific phages. The Lactobacillus strains selected as adjunct cultures for cheese making experiments reached 10⁸ cfu/g in soft cheeses at 7 d of ripening, whereas they reached 10⁹ cfu/g in semihard cheeses after 15 d of ripening. In both cheese varieties, the adjunct culture population remained at high counts during all ripening, in some cases overcoming or equaling primary starter. Overall, proximate composition of cheeses with and without added lactobacilli did not differ; however, some of the tested strains continued acidifying during ripening, which was mainly noticed in soft cheeses and affected overall quality of the products. The lactobacilli strains with low acidifying activity showed appropriate technological characteristics for their use as adjunct cultures in soft and semihard cheeses.