Kieran N. Kilcawley

Enzyme-modified cheese

Abstract This review highlights areas of interest in the production of enzyme-modified cheese. The demand for cheese flavours has increased due to consumer demand for a wider choice of convenience and low-fat products that possess cheese flavour. At present the best method for producing economic and consistent cheese flavours is through enzyme-modified cheese production. Essentially, the technology used to produce enzyme-modified cheese involves incubating cheese/curd with enzymes (proteinases, peptidases, lipases and esterases) in a slurry system under controlled conditions until the required flavour is reached. The flavour profile of enzyme modified cheese can be up to 30 times the intensity of natural cheese. A myriad of cheese flavours can be produced in this manner but a detailed understanding of the biochemistry of cheese flavours is required before production can be achieved on a consistent basis.

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.

Evaluation of two food grade proliposomes to encapsulate an extract of a commercial enzyme preparation by microfluidization.

The entrapment by microfluidization of a commercial enzyme extract (Debitrase DBP20) in liposomes using two food grade proliposome (C and S) preparations was studied. Liposomes obtained at a low microfluidization pressure (4000 psi) were distributed in a bimodal population of small (30-40 nm) and large vesicles (300-700 nm). The composition of the proliposome influenced entrapment efficiency and the repartition of the enzyme between the core and the surface of the liposome. More enzyme was associated with the liposomal surface and greater entrapment efficiencies (64%) were obtained for liposomes with the highest negative zeta potential (proliposome C). Increasing microfluidization pressure and increasing the number of passes through the microfluidizer resulted in losses in entrapment efficiency and enzyme activity, due to decreasing liposome size and enzyme denaturation. Entrapment efficiency was not influenced by external pH and enzyme activity was not adversely affected over storage for 18 days under the conditions evaluated.

Microbial Succession and Flavor Production in the Fermented Dairy Beverage Kefir

Traditional fermented foods represent relatively low-complexity microbial environments that can be used as model microbial communities to understand how microbes interact in natural environments. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein. In the process, the link between individual species, and associated pathways, with flavor compounds is revealed and several genes that could be responsible for the purported gut health-associated benefits of consuming kefir are identified. Ultimately, in addition to providing an important fundamental insight into microbial interactions, this information can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods. ABSTRACT Kefir is a putatively health-promoting dairy beverage that is produced when a kefir grain, consisting of a consortium of microorganisms, is added to milk to initiate a natural fermentation. Here, a detailed analysis was carried out to determine how the microbial population, gene content, and flavor of three kefirs from distinct geographic locations change over the course of 24-h fermentations. Metagenomic sequencing revealed that Lactobacillus kefiranofaciens was the dominant bacterial species in kefir during early stages of fermentations but that Leuconostoc mesenteroides became more prevalent in later stages. This pattern is consistent with an observation that genes involved in aromatic amino acid biosynthesis were absent from L. kefiranofaciens but were present in L. mesenteroides. Additionally, these shifts in the microbial community structure, and associated pathways, corresponded to changes in the levels of volatile compounds. Specifically, Acetobacter spp. correlated with acetic acid; Lactobacillus spp. correlated with carboxylic acids, esters and ketones; Leuconostoc spp. correlated with acetic acid and 2,3-butanedione; and Saccharomyces spp. correlated with esters. The correlation data suggest a causal relationship between microbial taxa and flavor that is supported by observations that addition of L. kefiranofaciens NCFB 2797 increased the levels of esters and ketones whereas addition of L. mesenteroides 213M0 increased the levels of acetic acid and 2,3-butanedione. Finally, we detected genes associated with probiotic functionalities in the kefir microbiome. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein and can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods. IMPORTANCE Traditional fermented foods represent relatively low-complexity microbial environments that can be used as model microbial communities to understand how microbes interact in natural environments. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein. In the process, the link between individual species, and associated pathways, with flavor compounds is revealed and several genes that could be responsible for the purported gut health-associated benefits of consuming kefir are identified. Ultimately, in addition to providing an important fundamental insight into microbial interactions, this information can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods. Author Video: An author video summary of this article is available.

A survey of the composition and proteolytic indices of commercial enzyme-modified Cheddar cheese

The compositional and proteolytic parameters in a range of commercial enzyme-modified Cheddar cheeses were quantified, with large variations evident between products from the same manufacturer and from different manufacturers. Analysis of the products indicated the use of cheese of varying fat content, exogenous protein and/or fat, emulsifying salts, flavour potentiators and bulking agents. Extensive proteolysis was a characteristic of these commercial products. Overall, production of enzyme-modified Cheddar cheese involves manipulation of both composition and proteolysis to generate products for specific applications.

Comparative and functional genomics of the Lactococcus lactis taxon; insights into evolution and niche adaptation

BackgroundLactococcus lactis is among the most widely studied lactic acid bacterial species due to its long history of safe use and economic importance to the dairy industry, where it is exploited as a starter culture in cheese production.ResultsIn the current study, we report on the complete sequencing of 16 L. lactis subsp. lactis and L. lactis subsp. cremoris genomes. The chromosomal features of these 16 L. lactis strains in conjunction with 14 completely sequenced, publicly available lactococcal chromosomes were assessed with particular emphasis on discerning the L. lactis subspecies division, evolution and niche adaptation. The deduced pan-genome of L. lactis was found to be closed, indicating that the representative data sets employed for this analysis are sufficient to fully describe the genetic diversity of the taxon.ConclusionsNiche adaptation appears to play a significant role in governing the genetic content of each L. lactis subspecies, while (differential) genome decay and redundancy in the dairy niche is also highlighted.

PROPERTIES OF COMMERCIAL MICROBIAL PROTEINASE PREPARATIONS

ABSTRACT Twenty three commercial microbial proteinase preparations derived from various Bacillus or Aspergillus spp. or from Rhizomucor niveus were assessed for proteolytic activity on azocasein at pH 5.5 or 7.0, or specificity on sodium caseinate at pH 5.5 and semi-quantitatively assessed for esterase, lipase, trypsin, chymotrypsin, general aminopeptidase, phosphatase and glycosidase activities using the API-ZYM system. Selected preparations were further assayed for peptidase, esterase and lipase activities at pH 7.0. The proteolytic activity of the Bacillus preparations was greater at pH 7.0, while that of the Aspergillus and Rhizomucor preparations was greater at pH 5.5. All the Bacillus preparations contained one of two main proteolytic activities, thought to be either bacillolysin or subtilisin. Most of the Aspergillus preparations contained the same proteinase, thought to be aspergillopepsin I, but two preparations appeared to contain a different unidentified proteinase. The proteolytic specificity of the Rhizomucor preparation was different from the Bacillus or Aspergillus preparations; thought to be rhizopuspepsin. According to the results of the API-ZYM system, all preparations contained enzyme activities in addition to their main proteolytic activity, with the Aspergillus and Rhizomucor preparations containing the highest levels and widest range of activities. Generally preparations derived from Aspergillus contained the highest level of general, proline and endopeptidase activities, with the Bacillus preparations conspicuous by the absence of general and proline-specific peptidase activities, while the Rhizomucor niveus preparation contained little or no general or endopeptidase activity. Esterase activity was found in all of the preparations evaluated, with only two Aspergillus preparations containing lipase activity.

Genetic, enzymatic and metabolite profiling of the Lactobacillus casei group reveals strain biodiversity and potential applications for flavour diversification

The Lactobacillus casei group represents a widely explored group of lactic acid bacteria, characterized by a high level of biodiversity. In this study, the genetic and phenotypic diversity of a collection of more than 300 isolates of the Lact. casei group and their potential to produce volatile metabolites important for flavour development in dairy products, was examined.

Effect of pasture versus indoor feeding systems on quality characteristics, nutritional composition, and sensory and volatile properties of full-fat Cheddar cheese

The purpose of this study was to investigate the effects of pasture-based versus indoor total mixed ration (TMR) feeding systems on the chemical composition, quality characteristics, and sensory properties of full-fat Cheddar cheeses. Fifty-four multiparous and primiparous Friesian cows were divided into 3 groups (n = 18) for an entire lactation. Group 1 was housed indoors and fed a TMR diet of grass silage, maize silage, and concentrates; group 2 was maintained outdoors on perennial ryegrass only pasture (GRS); and group 3 was maintained outdoors on perennial ryegrass/white clover pasture (CLV). Full-fat Cheddar cheeses were manufactured in triplicate at pilot scale from each feeding system in September 2015 and were examined over a 270-d ripening period at 8°C. Pasture-derived feeding systems were shown to produce Cheddar cheeses yellower in color than that of TMR, which was positively correlated with increased cheese β-carotene content. Feeding system had a significant effect on the fatty acid composition of the cheeses. The nutritional composition of Cheddar cheese was improved through pasture-based feeding systems, with significantly lower thrombogenicity index scores and a greater than 2-fold increase in the concentration of vaccenic acid and the bioactive conjugated linoleic acid C18:2 cis-9,trans-11, whereas TMR-derived cheeses had significantly higher palmitic acid content. Fatty acid profiling of cheeses coupled with multivariate analysis showed clear separation of Cheddar cheeses derived from pasture-based diets (GRS or CLV) from that of a TMR system. Such alterations in the fatty acid profile resulted in pasture-derived cheeses having reduced hardness scores at room temperature. Feeding system and ripening time had a significant effect on the volatile profile of the Cheddar cheeses. Pasture-derived Cheddar cheeses had significantly higher concentrations of the hydrocarbon toluene, whereas TMR-derived cheese had significantly higher concentration of 2,3-butanediol. Ripening period resulted in significant alterations to cheese volatile profiles, with increases in acid-, alcohol-, aldehyde-, ester-, and terpene-based volatile compounds. This study has demonstrated the benefits of pasture-derived feeding systems for production of Cheddar cheeses with enhanced nutritional and rheological quality compared with a TMR feeding system.

The Use of Viable and Heat-shocked Lactobacillus helveticus DPC 4571 in Enzyme-Modified Cheese Production

The use of viable or attenuated Lactobacillus helveticus DPC 4571 for use in enzyme-modified cheese production was assessed. Optimal heat shocking conditions for attenuation of DPC 4571 were found to be 69°C for 25 sec. Enzyme-modified cheese was produced from an emulsion of pre-hydrolysed rennet curd, water, and butter fat. This substrate was heat-treated and inoculated with either an equivalent level of viable or attenuated cells of DPC 4571 and further incubated under controlled conditions. The heat-treated products produced using attenuated DPC 4571 had a preferred sensory character with strong cheesy savory notes, enhanced secondary proteolysis, and more key volatile flavor compounds than those produced with viable DPC 4571. However, prolonged incubation (>16 h) resulted in growth of advantageous enterococci, which adversely influenced the sensory profile.