Cheese Starter Research Articles

A high-throughput cheese manufacturing model for effective cheese starter culture screening

Cheese making is a process in which enzymatic coagulation of milk is followed by protein separation, carbohydrate removal, and an extended bacterial fermentation. The number of variables in this complex process that influence cheese quality is so large that the developments of new manufacturing protocols are cumbersome. To reduce screening costs, several models have been developed to miniaturize the cheese manufacturing process. However, these models are not able to accommodate the throughputs required for systematic screening programs. Here, we describe a protocol that allows the parallel manufacturing of approximately 600 cheeses in individual cheese vats each with individual process specifications. Protocols for the production of miniaturized Gouda- and Cheddar-type cheeses have been developed. Starting with as little as 1.7mL of milk, miniature cheeses of about 170mg can be produced and they closely resemble conventionally produced cheese in terms of acidification profiles, moisture and salt contents, proteolysis, flavor profiles, and microstructure. Flavor profiling of miniature cheeses manufactured with and without mixed-strain adjunct starter cultures allowed the distinguishing of the different cheeses. Moreover, single-strain adjunct starter cultures engineered to overexpress important flavor-related enzymes revealed effects similar to those described in industrial cheese. Benchmarking against industrial cheese produced from the same raw materials established a good correlation between their proteolytic degradation products and their flavor profiles. These miniature cheeses, referred to as microcheeses, open new possibilities to study many aspects of cheese production, which will not only accelerate product development but also allow a more systematic approach to investigate the complex biochemistry and microbiology of cheese making.

Phenotypic and genotypic characterization of lactobacilli from Churpi cheese

The aim of this study was to investigate the diversity of lactobacilli in Churpi cheese, a traditional variety of cheese made from yak milk. Thirty-five lactobacilli isolated from five different samples procured from different parts of Arunachal Pradesh (India) were analyzed by phenotypic and genotypic methods. The 16S rDNA sequencing for all the isolates was performed. Five different species of lactobacilli were isolated from Churpi cheese with the predominance of Lactobacillus paracasei followed by Lactobacillus plantarum. All the isolates were further evaluated for their technological properties such as citrate utilization, exopolysaccharide (EPS), bacteriocin, and acid production. Forty-three percent isolates were found positive for citrate utilization, while 14% were found good acid producer, and 11% for EPS production. Lactobacillus coryniformis was an uncommon species found in Churpi cheese. Isolates obtained in this study can be potentially used for the development of defined strain starter for Churpi cheese.

Phenotypic variation inLactococcus lactissubsp.lactisisolates derived from intestinal tracts of marine and freshwater fish

We compared phenotypic characteristics of Lactococcus lactis subsp. lactis derived from different sources including the intestinal tract of marine fish and freshwater fish, and cheese starter culture. In the phylogenetic analysis based on partial 16S rRNA gene nucleotide sequences (1371 bp), freshwater fish-, marine fish- and cheese starter culture-derived strains were identical to that of L. lactis subsp. lactis previously reported. Fermentation profiles determined using the API 50 CH system were similar except for fermentation of several sugars including l-arabinose, mannitol, amygdalin, saccharose, trehalose, inulin and gluconate. The strains did have distinct levels of halotolerance: marine fish-derived strains > cheese starter-derived strain > freshwater fish-derived isolate. Lactococcus lactis subsp. lactis showed extensive diversity in phenotypic adaptation to various environments. The phenotypic properties of these strains suggested that L. lactis subsp. lactis strains from fish intestine have additional functions compared with the cheese starter-derived strain that has previously described. The unique phenotypic traits of the fish intestinal tract-derived L. lactis subsp. lactis might make them useful as a probiotics in aquaculture, and contribute to the development of functional foods and novel food additives, since the strains derived from fish intestines might have additional functions such as antibacterial activity.

Allele Variation of Seven Housekeeping Genes in Twenty-One Dairy Enterococci

ABSTRACTA collection of twenty-one dairy Enterococci isolated from cheese starters was subjected to phylogenetic analysis with a recently proposed new molecular method called multilocus single-strand conformation polymorphism (MSSCP). The obtained set of allele profiles related to the determined loci was presented through their frequencies. A classification analysis was applied in order to obtain the variation between the alleles studied.Concerning the clustering of some of the species after the applied classification the allele variation assessment defines two major groups corresponding to Ent. faecium and Ent. faecalis. The applied method for phylogeic analysis through UPGMA has good discriminative power between the strains of the studied species.

Characterization of Bioactive Peptides with Calcium-Binding Activity Released by Specially Designed Cheese Starter

ABSTRACTA total number of 210 strains of lactobacilli were isolated from different sources—homemade yoghurts and cheeses, plants and human fecal samples. The species affiliation of the strains was analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA) followed by 16S rDNA sequencing. The strain differentiation was obtained by methods of Pulsed Field Gel Electrophoresis (PFGE) and Amplified Fragment Length Polymorphism (AFLP), which led to strain-specific genetic profiles. The general proteolytic activity for every strain was measured through derivatization of low-molecular weight peptide matter with ortho-phtalic aldehyde (OPA) and the strains with the best proteolytic activity were selected. Special peptidase activities were studied applying colored substrates. The ability of the selected strains to produce peptides possessing calcium and magnesium binding properties was evaluated by help of ion-selective electrode, measuring the concentration of the Ca ions non-bonded by peptides. Peptides with Ca binding effect from the strains demonstrating the highest Ca attraction were subjected to fractionating on different HPLC columns. Such peptides were isolated, purified and sequenced. Finally, two strains which were capable to produce bioactive peptides with the strongest Ca- and Mg-binding activities were selected for development of cheese starters. After pilot production of functional cheeses with the developed starters the functional effect of bioactive peptides was confirmed during the cheese ripening.

Detection and Preliminary Characterization of a Blis Produced by Enterococcus Strain Isolated from Cheese Starter

ABSTRACTA collection of newly isolated strains identified as Enterococcus was screened for bacteriocin activity with molecular approaches. It was found that one of the tested strains displayed a broad spectrum activity, mainly against Gram-positive bacterial species. The agent was proven to be of protein nature. High temperature treatment revealed the relatively thermolabile properties of the molecule. It has comparatively high molecular weight for a bacteriocin, which was determined with NNWL filter units.

Comparison of the effects of various attenuation methods on cell permeability and accessibility of intracellular enzymes in Lactococcus lactis strains

Three Lactococcus lactis cheese starter cultures were exposed to various cell attenuation methods including sonication, chemical treatments, heat-shock and freeze-shock. Treated cells were subsequently assessed for changes in cell permeability and accessibility to the intracellular peptidases Pep X and Pep N. Generally, for all three strains, as the duration of sonication increased, the levels of intracellular peptidases in the cell free extract increased. For the other treatments, cell pellets of strains were prepared and treated with isopropyl alcohol (IPA), sodium dodecyl sulphate (SDS), hexadecyltrimethylammonium bromide (CTAB), heated over the range 45–65 °C or freeze-shocked using liquid nitrogen. The effects of various treatments on cell permeability were assessed using flow cytometry after staining with the Live/Dead ® BacLight™ reagent kit. Treatment of cell pellets with CTAB generated a unique and highly permeabilized sub-population having the highest levels of accessible intracellular enzyme activity. Treatment with SDS also resulted in highly permeabilized cells and enhanced accessibility to Pep X or Pep N. The findings in this study are significant for the production of permeabilized L. lactis cell fractions having enhanced accessibility to intracellular enzymes.

Genomics of dairy fermentations.

Microbes are used in the process of making industrial and artisanal fermented dairy products, such as cheese, yogurt, sour cream and fermented milks (Fig. 1). These microbes are predominately lactic acid bacteria (LAB), such as lactococci, lactobacilli and streptococci. For quality and consistency, industrial production requires the use of starter cultures, which are very carefully created, cultivated and maintained (Fig. 2). What happens in the fermentation process? Milk sugars (mainly lactose) are fermented with the major final product being lactic acid. Lactic acid not only inhibits the out‐growth of other organisms but also lowers the pH of the food product. Taste and texture, the feeling of food in your mouth, is also important. Lactic acid bacteria make the specific end‐products that impart flavour and modify the texture of the final product. Cheese production makes use of predominantly Lactococcus lactis. It is the major component of cheese starter cultures and, as the worldwide cheese market is huge, it is one of the most important microbes for the food industry. Several of the important functions for fermentation are encoded on conjugative plasmids in these bacteria, among them lactose metabolism and the breakdown of milk proteins during cheese production (Siezen et al., 2005; Shearman et al., 2008). The lactobacilli are also important players in dairy fermentations with Lactobacillus bulgaricus mainly used in yoghurt manufacture, together with Streptococcus thermophilus. This use of microbial consortia adds yet another degree of complexity to an already complex production process. Figure 1 From milk to fermented dairy product. Figure 2 Starter cultures for dairy fermentations. There are now over 20 genomes of LAB published and annotated, providing insight into their metabolic capabilities, as reviewed in (Pfeiler and Klaenhammer, 2007; Mayo et al., 2008). Comparing these genomes for shared or unique genotypes is a start, but the world of dairy fermentation is not content just with comparison. The real questions that are being asked are: what makes my yogurt or cheese different, and how can I develop new flavours, textures and products? This is increasingly being investigated by natural diversity analysis of microbes, and in situ omics measurements in dairy products. These and many other studies were reported at the 9th Symposium on LAB: Health, Evolution and Systems Biology, held in September 2008 (http://www.lab9.nl). Here we highlight some of the latest developments in genomics in these areas.

Production of Hard-Type Cheese Using Free or Immobilized Freeze-Dried Kefir Cells as a Starter Culture

This study provides a contribution to hard-type cheese starter culture production through the use of a freeze-dried culture in the ripening of hard-type cheeses. The effect of initial cell concentration, ripening temperature, and cell immobilization of kefir on the degree of openness, mold spoilage, microbial associations, physicochemical characteristics, and aroma-related compounds was studied. Use of kefir starter cultures resulted in cheese with an increased shelf life and resistance to spoilage as compared to control cheeses without kefir inoculants. Furthermore, the freeze-dried kefir culture improved aroma, taste, and texture characteristics while increasing the degree of openness in comparison to traditional hard-type cheese products. The kefir culture resulted in an increase in counts of total aerobic bacteria, yeasts and molds, lactococci, and lactobacilli until the 15th day of ripening. From then on, only lactobacilli counts increased, reaching levels up to 9.17 log CFU/g in cheeses ripened at 5 degrees C using freeze-dried kefir cells immobilized on casein. SPME-GC/MS analysis revealed major differences in volatile composition, especially with regard to alcohols (up to 75%), carbonyl compounds (up to 75%), and esters (up to 64%) between cheeses made with kefir cells and cheeses made without kefir inoculants.

Development of biocontrol agents from food microbial isolates for controlling post-harvest peach brown rot caused by Monilinia fructicola

An unconventional strategy of screening food microbes for biocontrol activity was used to develop biocontrol agents for controlling post-harvest peach brown rot caused by Monilinia fructicola. Forty-four microbial isolates were first screened for their biocontrol activity on apple fruit. Compared with the pathogen-only check, seven of the 44 isolates reduced brown rot incidence by > 50%, including four bacteria: Bacillus sp. C06, Lactobacillus sp. C03-b and Bacillus sp. T03-c, Lactobacillus sp. P02 and three yeasts: Saccharomyces delbrueckii A50, S. cerevisiae YE-5 and S. cerevisiae A41. Eight microbial isolates were selected for testing on peaches by wound co-inoculation with mixtures of individual microbial cultures and conidial suspension of M. fructicola. Only two of them showed significant biocontrol activity after five days of incubation at 22 °C. Bacillus sp. C06 suppressed brown rot incidence by 92% and reduced lesion diameter by 88% compared to the pathogen-only check. Bacillus sp.T03-c reduced incidence and lesion diameter by 40% and 62%, respectively. The two isolates were compared with Pseudomonas syringae MA-4, a biocontrol agent for post-harvest peach diseases, by immersing peaches in an aliquot containing individual microbial isolates and the pathogen conidia. Treatments with isolates MA-4, C06 and T03-c significantly controlled brown rot by 91, 100, and 100% respectively. However, only isolates MA-4 and C06 significantly reduced brown rot by 80% and 15%, respectively when bacterial cells alone were applied. On naturally infected peaches, both the bacterial culture and its cell-free filtrate of the isolate C06 significantly controlled peach decay resulting in 77 and 90% reduction, respectively, whereas the treatment using only the bacterial cells generally had no effect. Isolate C06 is a single colony isolate obtained from a mesophilic cheese starter, and has been identified belonging to Bacillus amyloliquefaciens. The results have clearly demonstrated that isolate C06 has a great potential for being developed into a biocontrol agent.

CHARACTERIZATION OF THE MICROFLORA ISOLATED FROM QUESO FRESCO MADE FROM RAW AND PASTEURIZED MILK*

ABSTRACT This study characterized the microflora present in four Queso Fresco‐style cheeses made from raw milk (RM) and two cheeses made from pasteurized milk (PM) obtained directly from six different producers in Mexico. Microbial isolates were initially analyzed on 8 different selective media and further analyzed by 16S rRNA gene sequence analysis. Sequence analysis identified Lactococcus lactis ssp. lactis as an isolate growing on M17 agar in four cheese samples. L. lactis ssp. lactis was the only lactic acid bacterium identified which is traditionally used as a cheese starter culture. Sequence analysis of isolates from MRS medium supplemented with vancomycin identified Leuconostoc mesenteroides in all six cheese samples. A high number of coliforms, enterococci and coagulase‐positive staphylococci were identified in all RM cheeses. The numbers of total coliform species were lower in both PM samples (by 2 log10), but the numbers of staphylococci were lower in only one sample (by 3 log10). The numbers of enterococci were high in both PM samples, and sequence analysis identified Enterococcus faecium as the only enterococcal species isolated from all six cheeses. These results suggest that L. lactis ssp. lactis, L. mesenteroides and E. faecium are species that may contribute to the organoleptic qualities of Queso Fresco‐style cheeses analyzed in this study.PRACTICAL APPLICATIONSThis work was performed to define the natural microflora present in traditional‐style Queso Fresco in order to identify bacterial species that may contribute to the organoleptic qualities of the cheese. Identification and enumeration of the “generally regarded as safe” (GRAS) lactic acid bacteria may allow for the development of new culture blends for the production of Queso Fresco using pasteurized milk, while maintaining the desired qualities of the raw milk varieties. In addition, bacteria recovered from the raw milk cheeses may be novel strains offering advantages over commercially available starter and adjunct LAB currently being used to make Queso Fresco. The work also identified potential pathogens and spoilage bacteria associated with traditional‐style Queso Fresco, stressing the importance in developing a desirable pasteurized milk cheese.

Evaluation of Aroma Generation of Lactococcus lactis with an Electronic Nose and Sensory Analysis

There is an increased interest in exploring the potential of new Lactococcus lactis strains isolated from different natural ecosystems for the production of aroma compounds. Thus, the objective of this study was to screen the aroma generation of Lactococcus lactis strains isolated from different sources by an electronic nose and sensory evaluation for their potential use in starter cheese cultures. Twenty-three strains of Lactococcus lactis were isolated from dairy sources such as artisanal raw-milk cheeses, nondairy sources, and commercial starter cultures (industrial). All the strains were assessed for their ability to produce aromas by an electronic nose and sensory analysis after their incubation in milk. Some phenotypic characteristics of technological importance such as lactose fermentation, proteolytic activity, and citrate utilization were also evaluated. Lactococcus lactis strains showed clear phenotypic differences related to their isolation source. Strains isolated from raw-milk dairy products or commercial starter cultures presented faster lactose fermentation and proteolytic activity than those presented by strains isolated from nondairy sources. Additionally, strains isolated from dairy and nondairy sources presented better citrate utilization than strains isolated from commercial dairy starters. On the other hand, there was not a clear relationship between the source of isolation and the ability of lactococci strains to produce aroma. Principal components analysis of electronic nose data revealed 4 distinctive groups based on aroma profiles. Additionally, odor intensity scores (yogurt-like and Fresco cheese-like) for these 4 groups revealed the nature of their differences. In general, strains from dairy products presented intense yogurt-like and Fresco cheese-like aromas, with the latter being the most intense for one specific strain. On the other hand, the majority of wild strains from nondairy sources presented a stronger yogurt-like aroma, whereas industrial strains presented low intensity for both aroma descriptors. Additionally, an aroma potentiation effect was observed when mixtures of 2 lactococci strains isolated from different sources acted together.

Isolation of halotolerant Lactococcus lactis subsp. lactis from intestinal tract of coastal fish

We isolated lactic acid bacteria from the intestinal tract of the pufferfish Takifugu niphobles caught in Shimoda, Shizuoka, Japan by using MRS broth prepared with 50% seawater. Additional screening was carried out using phenotypic tests such as Gram staining, cell morphology, catalase, oxidase and fermentation of glucose. Subsequently 227 isolates screened by the phenotypic tests were subjected to species-specific PCR for Lactococcus lactis, resulting in four positive isolates. The 16S rRNA gene sequences from three isolates were highly similar to that of L. lactis subsp. lactis (DNA database accession number M58837), while that of one isolate was identical to that of Leuconostoc mesenteroides ( AB023246). These isolates were characterized by API 50 CH for carbohydrate fermentation and other phenotypic criteria for salt tolerance, and the characteristics were compared with those of L. lactis subsp. lactis from a cheese starter culture. The carbohydrate fermentation profiles of these isolates were characteristic of L. lactis subsp. lactis strains, whereas the tolerance of these isolates to salt was higher than that of L. lactis subsp. lactis from the cheese starter culture: the new L. lactis isolates showed high salt tolerance in MRS-agar plates containing 200% seawater or 6% sodium chloride. This is the first report of the isolation of halotolerant strains of L. lactis subsp. lactis from a marine environment.

Biogenic amine-forming microbial communities in cheese

The aim of this study was to screen two cheese starter cultures and cheese-borne microbial communities with the potential to produce biogenic amines in cheese during ripening. Bacteria of the genera Enterococcus and Lactobacillus and coliform bacteria were isolated from Dutch-type semi-hard cheese at the beginning of the ripening period. Statistically significant counts of bacterial isolates were screened for the presence of specific DNA sequences coding for tyrosine decarboxylase (tyrDC) and histidine decarboxylase (hDC) enzymes. The PCR analysis of DNA from 14 Enterococcus and 3 Lactobacillus isolates confirmed the presence of the targetted DNA sequences. Simultaneously, 13 tyrDC- and 3 hDC-positive isolates were grown in decarboxylase screening medium and this was followed by HPLC analysis of the produced tyramine and histamine. Conventional and molecular taxonomic analyses of the above-mentioned isolates identified the following species: Enterococcus durans (7 strains), Enterococcus faecalis (3 strains), Enterococcus faecium (1 strain), Enterococcus casseliflavus (3 strains), Lactobacillus curvatus (1 strain), Lactobacillus lactis (1 strain) and Lactobacillus helveticus (1 strain). All of the above Enterococcus and two of the Lactobacillus strains originated from contaminating microbial communities. The L. helveticus strain, which was tyrosine decarboxylase-positive and exhibited tyramine production, originated from starter culture 1 used for cheese production. Comparison of partial tyrDC sequences of positive Enterococcus isolates revealed 89% sequence similarity, and that of hDC-positive Lactobacillus isolates revealed 99% sequence similarity.

Growth stimulation of a proteinase positive Lactococcus lactis strain by a proteinase negative Lactococcus lactis strain

Lactococcus lactis AMP15/pAMP31(D471R) is a proteinase negative, lactose negative strain with a modified oligopeptide transport system, and potential as a debittering agent due to its efficient utilization of hydrophobic peptides. Five wild L. lactis strains of dairy origin, which produced cheeses of high flavour quality, were cocultured with L. lactis AMP15/pAMP31(D471R) in an attempt to select adequate combinations of strains for use as defined cheese starters with potential debittering ability. Four of these strains, L. lactis B6, K16, M21 and P21, inhibited growth of L. lactis AMP15/pAMP31(D471R) at a level of 10 6 to 10 7 cfu mL − 1 after 24 h of incubation, even though production of bacteriocin-like compounds could only be proven for L. lactis M21. When L. lactis AMP15/pAMP31(D471R) was cocultured with the fifth strain, L. lactis N22, its growth was significantly ( P < 0.001) inhibited whereas growth of L. lactis N22 was significantly stimulated. The nature of the interaction was studied and it was established that L. lactis N22 is auxotrophic for folate, a compound produced and excreted by L. lactis AMP15/pAMP31(D471R).

Galactose Metabolism and Capsule Formation in a Recombinant Strain of Streptococcus thermophilus with a Galactose-Fermenting Phenotype

The capsule-producing, galactose-negative Streptococcus thermophilus MR-1C strain was first transformed with a low-copy plasmid containing a functional galK gene from Streptococcus salivarius to generate a recombinant galactose-fermenting Strep. thermophilus strain named MR-AAC. Then, we compared the functional properties of Strep. thermophilus MR-AAC with those of the parent MR-1C strain when used as starter for fermented products and cheese. In lactose-supplemented laboratory medium, MR-AAC metabolized galactose, but only when the amount of lactose was less than 0.1% (wt/vol). After 7h of fermentation, the medium was almost depleted of galactose. The parent strain, MR-1C, showed the same pattern, except that the concentration of galactose decreased by only 25% during the same period. It was found that, during milk fermentation and Mozzarella cheese production, the galactose-fermenting phenotype was not expressed by MR-AAC and this strain expelled galactose into the medium at a level similar to the parent MR-1C strain. In milk and in lactose-supplemented medium, capsular exopolysaccharide production occurred mainly during the late exponential phase and the stationary growth phase with similar kinetics between MR-1C and MR-AAC.

MODEL OF THE CONTINUOUS PREFERMENTATION PROCESS IN CHEESE MANUFACTURE AND STABILITY ANALYSIS OF STEADY STATE

ABSTRACT The present study focused on the continuous prefermentation process affecting curd formation, and the subsequent pH changes in the first stage of cheese manufacture. Two typical continuous prefermentation systems in cheese manufacture were analyzed based on a kinetic model and the process control theory. A previously proposed mathematical model, which considers the effect of pH, adequately simulated the dynamic behavior of pH in the two continuous prefermentation processes. In the stability analysis of steady state using the Routh–Hurwitz criterion, we found that the continuous feed of a cheese starter culture prevented wash‐out and stabilized the system independent of the fraction of seed feed rate. When no cheese starter culture was fed, the stabilities of steady states were dependent on the partial differentials of the maximum specific growth rate to lactic acid concentration. The feeding of cheese starter culture was effective to improve prefermentation process performance.

Production of γ-aminobutyric acid by Streptococcus salivarius subsp. thermophilus Y2 under submerged fermentation

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, has several well-known physiological functions and has been applied to the production of many drugs and functional foods. The technology of GABA production via submerged fermentation by Streptococcus salivarius subsp. thermophilus Y2 was investigated in this paper. It indicated that the GABA production was related to the biochemical characteristics of glutamate decarboxylase (GAD) of S. salivarius subsp. thermophilus Y2. After 24 h of fermentation at 37 degrees C, which is the suitable culture conditions for GAD-production, then the culture condition were adjusted to the optimal temperature (40 degrees C) and pH (4.5) for the GAD reaction activity in biotransformation of cells and pyridoxal 5'-phosphate (0.02 mmol/l) were added to the broth at the 48 h, the GABA production was increased up to 1.76-fold, reaching 7984.75 +/- 293.33 mg/l. The strain shows great potential use as a starter for GABA-containing yoghurt, cheese and other functional fermented food productions.

Avoidance of medicines residues in milk: an update

MEDICINES residues in milk continue to remain a problem for the dairy industry, with failures running at a fairly constant level of 0·2 per cent of bulk supply despite a...

Characterisation of the non-starter lactic acid bacteria (NSLAB) of Gruyère PDO cheese

The diversity of non-starter lactic acid bacteria (NSLAB) of Swiss Gruyere cheese was studied in three factories over a six-month period. A total of 1099 isolates of NSLAB from raw milk, skimmilk and Gruyere cheese were characterised at both the species and strain level. Over 90% of the isolates were either Lactobacillus casei or L. rhamnosus, and a combined total of 45 genotypes were found. The genotypes from the three factories differed from one another and varied slowly over the six-month period. At one factory, samples from the corresponding raw milk, skimmilk and cheese culture were also analysed. No NSLAB were found in the cheese starter culture. On the con- trary, raw milk contained the largest number of different genotypes. The genotypes found in the cheese all came from raw milk. non-starter lactic acid bacteria / NSLAB / L. casei / L. rhamnosus / Gruyere cheese