Enhanced nutty flavor formation in cheddar cheese made with a malty Lactococcus lactis adjunct culture

Nutty flavor in Cheddar cheese is desirable, and recent research demonstrated that 2- and 3-methyl butanal and 2-methyl propanal were primary sources of nutty flavors in Cheddar. Because malty strains of Lac-tococcus lactis (formerly Streptococcus lactis var. malti-genes) are characterized by the efficient production of these and other Strecker aldehydes during growth, this study investigated the influence of a malty L. lactis adjunct culture on nutty flavor development in Cheddar cheese. Cheeses made with different adjunct levels (0, 10⁴ cfu/mL, and 10⁵ cfu/mL) were ripened at 5 or 13°C and analyzed after 1 wk, 4 mo, and 8 mo by a combination of instrumental and sensory methods to characterize nutty flavor development. Cheeses ripened at 13°C developed aged flavors (brothy, sulfur, and nutty fla-vors) more rapidly than cheeses held at 5°C. Additionally, cheeses made with the adjunct culture showed more rapid and more intense nutty flavor development than control cheeses. Cheeses that had higher intensities of nutty flavors also had a higher concentration of 2/3-methyl butanal and 2-methyl propanal compared with control cheeses, which again confirmed that these compounds are a source of nutty flavor in Cheddar cheese. Results from this study provide a simple methodology for cheese manufacturers to obtain consistent nutty flavor in Cheddar cheese.     

Nonstarter lactic acid bacteria biofilms and calcium lactate crystals in Cheddar cheese

A sanitized cheese plant was swabbed for the presence of nonstarter lactic acid bacteria (NSLAB) biofilms. Swabs were analyzed to determine the sources and microorganisms responsible for contamination. In pilot plant experiments, cheese vats filled with standard cheese milk (lactose:protein = 1.47) and ultrafiltered cheese milk (lactose:protein = 1.23) were inoculated with Lactococcus lactis ssp. cremoris starter culture (8 log cfu/mL) with or without Lactobacillus curvatus or Pediococci acidilactici as adjunct cultures (2 log cfu/mL). Cheddar cheeses were aged at 7.2 or 10°C for 168 d. The raw milk silo, ultrafiltration unit, cheddaring belt, and cheese tower had NSLAB biofilms ranging from 2 to 4 log cfu/100 cm². The population of Lb. curvatus reached 8 log cfu/g, whereas P. acidilactici reached 7 log cfu/g of experimental Cheddar cheese in 14 d. Higher NSLAB counts were observed in the first 14 d of aging in cheese stored at 10°C compared with that stored at 7.2°C. However, microbial counts decreased more quickly in Cheddar cheeses aged at 10°C compared with 7.2°C after 28 d. In cheeses without specific adjunct cultures (Lb. curvatus or P. acidilactici), calcium lactate crystals were not observed within 168 d. However, crystals were observed after only 56 d in cheeses containing Lb. curvatus, which also had increased concentration of d(−)-lactic acid compared with control cheeses. Our research shows that low levels of contamination with certain NSLAB can result in calcium lactate crystals, regardless of lactose:protein ratio.     

Response of two Salmonella enterica strains inoculated in model cheese treated with high hydrostatic pressure

The aim of this work was to determine the response to high hydrostatic pressure and the ability for survival, recovery, and growth of 2 strains of Salmonella enterica (Salmonella enteritidis and Salmonella typhimurium) inoculated in a washed-curd model cheese produced with and without starter culture. Inoculated samples were treated at 300 and 400 MPa for 10 min at room temperature and analyzed after treatment and after 1, 7, and 15 d of storage at 12° C to study the behavior of the Salmonella population. Cheese samples produced with starter culture and treated at 300 and 400 MPa showed maximum lethality; no significant differences in the baroresistant behavior of both strains were detected. Nevertheless, when starter culture was not present, the maximum lethality was only observed in cheese samples treated at 400 MPa, in the case of S. enteritidis. Ability to repair and grow was not observed in model cheese produced with starter culture and cell counts of treated samples decreased after 15 d of storage at 12° C. In cheese produced without starter culture, Salmonella cells showed the ability to repair and grow during the storage period, reaching counts over 3 log₁₀ (cfu/mL) in both applied treatments and serotypes. These results suggest that high hydrostatic pressure treatments are effective to reduce Salmonella population in this type of cheese, but the presence of the starter culture affects the ability of this microorganism to repair and grow during the storage period.     

Survival of Listeria monocytogenes introduced as a post-aging contaminant during storage of low-salt Cheddar cheese at 4, 10, and 21°C

Traditional aged Cheddar cheese does not support Listeria monocytogenes growth and, in fact, gradual inactivation of the organism occurs during storage due to intrinsic characteristics of Cheddar cheese, such as presence of starter cultures, salt content, and acidity. However, consuming high-salt (sodium) levels is a health concern and the dairy industry is responding by creating reduced-salt cheeses. The microbiological stability of low-salt cheese has not been well documented. This study examined the survival of L. monocytogenes in low-salt compared with regular-salt Cheddar cheese at 2 pH levels stored at 4, 10, and 21°C. Cheddar cheeses were formulated at 0.7% and 1.8% NaCl (wt/wt) with both low and high pH and aged for 10 wk, resulting in 4 treatments: 0.7% NaCl and pH 5.1 (low salt and low pH); 0.7% NaCl and pH 5.5 (low salt and high pH); 1.8% NaCl and pH 5.8 (standard salt and high pH); and 1.8% NaCl and pH 5.3 (standard salt and low pH). Each treatment was comminuted and inoculated with a 5-strain cocktail of L. monocytogenes at a target level of 3.5 log cfu/g, then divided and incubated at 4, 10, and 21°C. Survival or growth of L. monocytogenes was monitored for up to 90, 90, and 30 d, respectively. Listeria monocytogenes decreased by 0.14 to 1.48 log cfu/g in all treatments. At the end of incubation at a given temperature, no significant difference existed in L. monocytogenes survival between the low and standard salt treatments at either low or high pH. Listeria monocytogenes counts decreased gradually regardless of a continuous increase in pH (end pH of 5.3 to 6.9) of low-salt treatments at all study temperatures. This study demonstrated that post-aging inoculation of L. monocytogenes into low-salt (0.7%, wt/wt) Cheddar cheeses at an initial pH of 5.1 to 5.5 does not support growth at 4, 10, and 21°C up to 90, 90, and 30 d, respectively. As none of the treatments demonstrated more than a 1.5 log reduction in L. monocytogenes counts, the need for good sanitation practices to prevent post-manufacturing cross contamination remains.     

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.     

Influence of adjunct use and cheese microenvironment on nonstarter bacteria in reduced-fat cheddar-type cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7 degrees C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 10(4) cfu/ml did not completely suppress "wild" NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.     

Differentiation of intracellular peptidases of starter and adjunct cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Selection of starter and adjunct cultures is important to minimize bitterness of Cheddar and Gouda cheeses. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry may be useful for rapid screening of cheese cultures for propensity to produce bitter cheese. The objective of this study was to demonstrate the application of MALDI-TOF for differentiating intracellular peptidase activities of starter and adjunct cultures on β-CN f193-209 under simulated cheese condition. Bovine β-casein was incubated with chymosin in 9.55 g/l citrate buffer (pH 5.4, 40 g/l sodium chloride) at 30°C for 24 h, followed by incubation with cell-free extract (CFE) of starter or adjunct culture. Mixed strains of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris designated as 56 and 105 were the sources of nonbitter and bitter starter cultures, respectively. Lactobacillus helveticus WSU-19 and W900R represented adjunct cultures having high and low debittering activities, respectively. The degradation pattern of β-CN f193-209 by CFE of WSU-19 indicates general aminopeptidase and endopeptidase activities, while degradation of the peptide by CFE of W900R, 56, and 105 are mainly from endopeptidase activity. The rates of β-CN f193-209 hydrolysis by CFE of WSU-19, W900R, 56, and 105 are 6.90, 0.38, 0.39, and 0.23 mg/l per h, respectively.     

Invited review: Lactobacillus helveticus—A thermophilic dairy starter related to gut bacteria

The strain Lactobacillus helveticus DPC4571 has emerged as a promising flavor adjunct culture for Cheddar cheese given that it is consistently associated with improved flavor. The availability of the complete genome sequence of Lb. helveticus DPC4571 has enabled the search for the presence or absence of specific genes on the genome, in particular those of technological interest. Indeed, this analysis has facilitated a greater understanding into the functioning of lactic acid bacteria as a whole. The biochemical pathways of Lb. helveticus responsible for producing flavor compounds during cheese ripening are poorly understood but now with the availability of a complete genomic sequence are ripe for exploitation. Bioinformatic analysis of the genome of Lb. helveticus DPC4571 has revealed a plethora of genes with industrial potential including those responsible for key metabolic functions that contribute to cheese flavor development such as proteolysis, lipolysis, and cell lysis. In addition, it has been demonstrated that Lb. helveticus has the potential to produce bioactive peptides such as angiotensin converting enzyme inhibitory activity in fermented dairy products, demonstrating the therapeutic value of this species. A most intriguing feature of the genome of Lb. helveticus DPC4571 is the remarkable similarity in gene content with many intestinal lactobacilli, although originating from considerably different environments. Bioinformatic analysis demonstrated that 65 to 75% of genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. This review focuses on the isolation, characterization, and exploitation of the Lb. helveticus species with particular emphesis on taking into consideration recent genome sequence data for Lb. helveticus and other Lactobacillus species.     

The effect of sodium reduction with and without potassium chloride on the survival of Listeria monocytogenes in Cheddar cheese

Sodium chloride (NaCl) in cheese contributes to flavor and texture directly and by its effect on microbial and enzymatic activity. The salt-to-moisture ratio (S/M) is used to gauge if conditions for producing good-quality cheese have been met. Reductions in salt that deviate from the ideal S/M range could result in changing culture acidification profiles during cheese making. Lactococcus lactis ssp. lactis or Lc. lactis ssp. cremoris are both used as cultures in Cheddar cheese manufacture, but Lc. lactis ssp. lactis has a higher salt and pH tolerance than Lc. lactis ssp. cremoris. Both salt and pH are used to control growth and survival of Listeria monocytogenes and salts such as KCl are commonly used to replace the effects of NaCl in food when NaCl is reduced. The objectives of this project were to determine the effects of sodium reduction, KCl use, and the subspecies of Lc. lactis used on L. monocytogenes survival in stirred-curd Cheddar cheese. Cheese was manufactured with either Lc. lactis ssp. lactis or Lc. lactis ssp. cremoris. At the salting step, curd was divided and salted with a concentration targeted to produce a final cheese with 600 mg of sodium/100 g (control), 25% reduced sodium (450 mg of sodium/100 g; both with and without KCl), and low sodium (53% sodium reduction or 280 mg of sodium/100 g; both with and without KCl). Potassium chloride was added on a molar equivalent to the NaCl it replaced to maintain an equivalent S/M. Cheese was inoculated with a 5-strain cocktail of L. monocytogenes at different times during aging to simulate postprocessing contamination, and counts were monitored over 27 or 50 d, depending on incubation temperature (12 or 5°C, respectively). In cheese inoculated with 4 log₁₀ cfu of L. monocytogenes/g 2 wk after manufacture, viable counts declined by more than 3 log₁₀ cfu/g in all treatments over 60 d. When inoculated with 5 log₁₀ cfu/g at 3 mo of cheese age, L. monocytogenes counts in Cheddar cheese were also reduced during storage, but by less than 1.5 log₁₀ cfu/g after 50 d. However, cheese with a 50% reduction in sodium without KCl had higher counts than full-sodium cheese at the end of 50 d of incubation at 4°C when inoculated at 3 mo. When inoculated at 8 mo postmanufacture, this trend was only observed in 50% reduced sodium with KCl, for cheese manufactured with both cultures. This enhanced survival for 50% reduced-sodium cheese was not seen when a higher incubation temperature (12°C) was used when cheese was inoculated at 3 mo of age and monitored for 27 d (no difference in treatments was observed at this incubation temperature). In the event of postprocessing contamination during later stages of ripening, L. monocytogenes was capable of survival in Cheddar cheese regardless of which culture was used, whether or not sodium had been reduced by as much as 50% from standard concentrations, or if KCl had been added to maintain the effective S/M of full-sodium Cheddar cheese.     

Production of fresh Cheddar cheese curds with controlled postacidification and enhanced flavor

Cheddar cheese in curd form is very popular in eastern Canada. It is retailed immediately after cheese manufacturing and can be maintained at room temperature for 24 h to provide better texture and mouthfeel. Subsequently, the cheese curds must be stored at 4°C. The shelf life is generally 3 d. In this study, Cheddar cheese curds were produced by adding a high diacetyl flavor-producing strain (Lactococcus diacetylactis) to a thermophilic-based starter. The objective was to achieve both postacidification stability to increase the shelf life and enhanced flavor. The addition of L. diacetylactis increased processing time but did not affect cheese composition or the evolution of proteolysis and texture. During cheese manufacturing, streptococci became the dominant microflora in all cheeses, whereas populations of Lactococcus cremoris and L. diacetylactis decreased. During cheese storage, viable counts of L. diacetylactis and Streptococcus thermophilus increased but the counts of L. cremoris decreased. During cheese manufacturing and storage, the concentrations of lactic acid and diacetyl increased rapidly in cheeses produced with L. diacetylactis. Citric acid and galactose contents remained high in cheese made without L. diacetylactis. Sensory evaluation indicated that cheeses containing the L. diacetylactis strain were more flavorful and also had less sourness and could be stored at 4°C for up to 7 d.     

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.     

Effect of exopolysaccharide produced by isogenic strains of Lactococcus lactis on half-fat Cheddar cheese

Fat-reduced cheeses often suffer from undesirable texture, flavor, and cooking properties. Exopolysaccharides (EPS) produced by starter strains have been proposed as a mechanism to increase yield and to improve the texture and cooking properties of reduced-fat cheeses. The objective of this work was to assess the influence of an exopolysaccharide on the yield, texture, cooking properties, and quality of half-fat Cheddar cheese. Two pilot-scale half-fat Cheddar cheeses were manufactured using single starters of an isogenic strain of Lactococcus lactis ssp. cremoris (DPC6532 and DPC6533) that differed in their ability to produce exopolysaccharide. Consequently, any differences detected between the cheeses were attributed to the presence of the exopolysaccharide. The results indicated that cheeses made with the exopolysaccharide-producing starter had an 8.17% increase in actual cheese yield (per 100 kg of milk), a 9.49% increase in moisture content, increase in water activity and water desorption rate at relative humidities ≤90%, significant differences in the cheeses microstructure, and a significant improvement in both textural and cooking properties, without negatively affecting the flavor profiles of the cheeses.     

基于快速成熟模型的藏灵菇发酵切达干酪挥发性风味物质分析

通过建立快速成熟干酪模型,采用固相微萃取法提取传统藏灵菇发酵的切达干酪模型与商品发酵剂制作的切达干酪模型中挥发性成分,并结合气相色谱-质谱联用技术和气相色谱-嗅闻技术对萃取成分进行鉴定,结果表明醇类和酯类是藏灵菇发酵切达干酪成熟过程中的主要风味物质。藏灵菇发酵切达干酪模型中风味物质的种类和含量都明显高于商业发酵剂制作的切达干酪模型,其中酯类物质的变化最为显著。感官评价和风味分析结果表明,藏灵菇发酵切达干酪模型中酯类和醇类物质种类和含量更为丰富,风味更强,水果香味更浓郁,还具有酒香味。     

Characterization of metabolic pathways for biosynthesis of the flavor compound 3-methylbutanal by Lactococcus lactis

3-Methylbutanal is a key volatile compound that imparts a nutty flavor to Cheddar cheese. Lactococcus lactis has been successfully applied as a starter to increase the level of 3-methylbutanal produced during the ripening of cheese. However, the mechanism of action and genetic diversity of this bacterium for 3-methylbutanal biosynthesis remains unclear. In this study, we investigated the association between the L. lactis genotype and phenotype in the biosynthesis of 3-methylbutanal via both direct and indirect pathways. Fourteen strains of L. lactis were screened for the capacity to produce 3-methylbutanal, and strain 408 (>140 μM) produced the highest among all tested strains, which exhibited both α-keto acid decarboxylase and α-ketoacid dehydrogenase activities. Furthermore, the results of a sodium meta-arsenite inhibition experiment showed that the 3-methylbutanal–producing capacities of each strain declined to various degrees. The kdcA gene, which encodes the direct pathway component α-ketoacid decarboxylase, was detected in 4 of the 14 strains, of which only strain 408 contained the full-length gene. We then characterized the genes associated with the indirect pathway by detecting the expression levels of the pdh gene cluster, ack, and pta, which were expressed at relatively higher levels in a high-yield strain than in a low-yield strain. As a result, these L. lactis strains were divided into 3 categories according to gene diversity, gene expression, and 3-methylbutanal production. The results of this study refine our knowledge of the genetic determinants of 3-methylbutanal biosynthesis in L. lactis and explain the effect of both synthesis pathways on 3-methylbutanal production.     

Characterization of Carnobacterium maltaromaticum LMA 28 for its positive technological role in soft cheese making

Carnobacterium maltaromaticum is a lactic acid bacterium isolated from soft cheese. The objective of this work was to study its potential positive impact when used in cheese technology. Phenotypic and genotypic characterization of six strains of C. maltaromaticum showed that they belong to different phylogenetic groups. Although these strains lacked the ability to coagulate milk quickly, they were acidotolerant. They did not affect the coagulation capacity of starter lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, used in dairy industry. The impact of C. maltaromaticum LMA 28 on bacterial flora of cheese revealed a significant decrease of Psychrobacter sp. concentration, which might be responsible for cheese aging phenomena. An experimental plan was carried out to unravel the mechanism of inhibition of Psychrobacter sp. and Listeria monocytogenes and possible interaction between various factors (cell concentration, NaCl, pH and incubation time). Cellular concentration of C. maltaromaticum LMA 28 was found to be the main factor involved in the inhibition of Psychrobacter sp. and L. monocytogenes.