Recent advances in cheese microbiology

Microorganisms are an essential component of cheeses and play important roles during both cheese manufacture and ripening. Both starter and secondary flora modify the physical and chemical properties of cheese, contributing and reacting to changes that occur during the manufacture and ripening of cheese. As the composition of microbial population changes under the influence of continuous shifts in environmental conditions and microorganisms interactions during manufacturing and ripening, the characteristics of a given cheese depend also on microflora dynamics. The microbiota present in cheese is complex and its growth and activity represent the most important, but the least controllable steps. In the past, research in this area was dependent on classical microbiological techniques. However, culture-dependent methods are time-consuming and approaches that include a culturing step can lead to inaccuracies due to species present in low numbers or simply uncultivable. Therefore, they cannot be used as a unique tool to monitor community dynamics. For these reasons approaches to cheese microbiology had to change dramatically. To address this, in recent years the focus on the use of culture-independent methods based on the direct analysis of DNA (or RNA) has rapidly increased. Application of such techniques to the study of cheese microbiology represents a rapid, sound, reliable, and effective way for the detection and identification of the microorganisms present in dairy products, leading to major advances in understanding this complex microbial ecosystem and its impact on cheese ripening and quality. In this article, an overview on the recent advances in the use of molecular methods for thorough analysis of microbial communities in cheeses is given. Furthermore, applications of culture-independent approaches to study the microbiology of two important raw-milk, long-ripened cheeses such as Grana Padano and Parmigiano Reggiano, are presented.

Chapter 15 - Microbiome Changes During Ripening

The dynamics of bacteria community of “Bola de Ocosingo” cheese, a Mexican artisanal raw milk cheese was investigated by high-throughput sequencing (454 pyrosequencing). Dairy samples (raw milk, curd, cheese at 50 and 110 days of ripening) were collected at dry (March-June) and rainy season (August-November) from three producers located in Chiapas, Mexico. In general, raw milk contained high bacterial diversity which was reduced throughout cheese manufacture. However, in two productions an important increase during cheese ripening was observed probably due to cross-contamination. Species such as Streptococcus thermophilus, Lactococcus lactis, Lactobacillus helveticus, L. delbrueckii and L. plantarum from which potential probiotic strains may be obtained, predominated during processing, varying its prevalence from one producer to another. Furthermore, low proportions of Escherichia coli/Shigella flexnerii were detected in almost all processes, however, could not be recovered by traditional methodology, indicating presence of non-cultivable cells. This work provides insights into bacteria communities of Bola de Ocosingo cheese for starter culture development, many of which are reported to provide health related benefits, and the usefulness of high-throughput sequencing to evidence cross-contamination during processing.

Enzyme Activities of Lactic Streptococci and Their Role in Maturation of Cheese: A Review

Many different kinds of cultures, enzymes, and methods are used during the production and ripening of a variety of cheese types. In this chapter, the importance, types, and applications of microbial cultures during cheese production are discussed. Moreover, an overview of the important role of enzymatic systems, either derived from these cultures or directly added to the milk fermentation, is presented. The main biochemical events including glycolysis, lipolysis, and proteolysis during cheese ripening are explained, focusing on their end products, which contribute to the development of the overall aroma of cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow’s milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

The aim of this paper was to develop a real-time PCR method targeting a gene sequence encoding a bacteriocin ABC transporter ATP-binding protein for the detection of Streptococcus thermophilus in cheese produced from raw cow milk. A real-time quantitative PCR assay was designated to identify and count S. thermophilus cells in ripened cheese. The developed real-time PCR primers and probe were highly specific for S. thermophilus CNRZ1066, CNRZ8232, LMD-9, LMG 18311, CNRZ 002 and CNRZ 03 but not for Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Leuconostoc spp., Enterococcus spp. that are phylogenetically very similar to S. thermophilus, and this is the reason why they are difficult to differentiate using culture-based methods. The real-time PCR allowed for quantification with a detection of 101–103 cfu/g of product. qPCR-standard curves were linear over seven log units down to 101 copies per reaction; PCR efficiencies ranged from 1.00 to 1.03. The newly developed qPCR method could be applied to specific detection and precise enumeration of S. thermophilus in raw milk and cheese.

Reverse transcription quantitative PCR revealed persistency of thermophilic lactic acid bacteria metabolic activity until the end of the ripening of Emmental cheese

Chapter 27 - Mold-Ripened and Raw Milk Cheeses: Production, Risks, and Benefits to Human Health

Cheese: Microbiology of Cheese

Branched-chain aldehydes are the primary compounds that contribute to the nutty flavor in cheddar cheese. Lactococcus lactis, which is often applied as primary starter culture, is a significant contributor to the nutty flavor of cheddar cheese due to its ability of conversion of BCAAs into branched-chain aldehydes. In the present study, we found that the regulatory role of CodY is crucial for the conversion. CodY acts as a pleiotropic transcriptional regulator via binding to various regulatory regions of key genes. The results presented valuable knowledge into the role of CodY on the regulation and biosynthetic pathway of branched-chain amino acids and the related aldehydes. Furthermore, it provided new insight for increasing the nutty flavor produced during the manufacture and ripening of cheese.

Cheese ripening causes intense proteolysis, particularly when the cheese contains starter cultures. Several studies have shown the presence of bioactive peptides in goat’s milk cheeses with antioxidant activity. Mexican goat cheeses’ peptide fractions were evaluated at different ripening stages. Additionally, they were correlated with their antioxidant activity. Proteolysis was measured in the acid-soluble nitrogen and non-protein nitrogen fractions using reverse-phase high-performance liquid chromatography. While the antioxidant activity in both nitrogenous fractions was determined using a 2,2-diphenyl-1-picrylhydrazyl solution. Analyzed cheeses showed peptides fraction in the retention time of 2.05, 18.36, and 50.11 min for acid-soluble fraction and non-protein protein nitrogen, and showed antioxidant activity from the first day of ripening to 73% discoloration in the DPPH solution at 55 ripening days. Obtained results suggested that ripened Mexican goat cheese had a DPPH radical scavenging activity related to peptides present originally in the milk or released by starter culture action during cheese ripening.

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it's necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese.

Cheese is a histamine-rich food and may contain biogenic amines formed during ripening process, due to degradation of proteins through proteolysis. Therefore, histamine producing bacteria and biogenic amines content were studied during ripening of ras cheese at refrigeration temperature 4±1oC for 10 weeks using HPLC. Three batches of Ras cheese were manufactured using Egyptian conventional method. From each batch, samples collected at zero, 2, 4, 6, 8 and 10 weeks after cheese manufacture. The obtained results revealed that Enterobacteriaceae and Enterococci had the same pattern during ripening of ras cheese with a significant decrease from (5.0x108±7.02x106 at zero day until reach 4.60x10±0.24x10 cfu/g at week 10 of storage), (from 3.46x105±3.17x104 at day zero until reach to 2.20x10±0.23x10 cfu/g at week 10 of storage), respectively. Lactobacilli count showed fluctuation during ripening period where the highest count recorded at 2nd and 10th week 4.40x105±0.25x105 and 4.40x105±0.15x105 cfu/g, respectively. Enterobacteriaceae, enterococci and lactobacilli producing histamine were identified. Investigated biogenic amines (Cadaverine, Histamine and Tyramine) significantly decrease during ripening of ras cheese while, Putrescine showing fluctuation during ripening. Therefore, to protect consumers from biogenic amines poisoning, factors that enhance biogenic amines formation during cheese processing should be controlled.

The effect of starters with a functional arginine deiminase pathway on cheese ripening and quality