Morphometric Indicators of Liver Acini of Deceased Newborns Depending on the Time of Death.

We studied morphometric changes in the liver acini of dead newborns depending on the duration of the postmortem period. Autopsy samples of the liver tissue from 49 dead newborns were divided into 7 groups depending on the time of death. Liver tissue samples were taken from the upper and lower areas of the liver in the supine position of newborns; paraffin sections were prepared and stained with hematoxylin and eosin. The morphometric analysis of histological preparations revealed a progressive decrease in the mean size of the liver plates (trabeculae) and, conversely, an increase in the area of sinusoids with increasing the duration of the postmortem period; these changes were due to the postmortem redistribution of the blood and autolysis processes. More significant changes were noted in acinar zone 3 of the lower part of the liver. The revealed intra-acinar features of postmortem changes should be taken into account for their differential diagnosis with pathological processes that developed during life, in particular, the signs of congestion and peliosis of the liver.

Coptis rhizome extract influence on Streptococcus pneumoniae through autolysin activation.

This study investigated the antibacterial properties of Coptis rhizome, a plant traditionally used for respiratory infections, against Streptoccus pneumonia (S. pneumoniae), for which there has been minimal empirical evidence of effectiveness. The study particularly examined autolysis, indirectly associated with antibacterial resistance, when using Coptis rhizome for bacterial infections. In our methodology, Coptis rhizome was processed with ethanol and distilled water to produce four different extracts: CRET30, CRET50, CRET70, and CRDW. The antibacterial activity of these extracts were tested through Minimum Inhibitory Concentration (MIC) assays, disk diffusion tests, and time-kill assays, targeting both standard (ATCC 49619) and resistant (ATCC 70067) strains. The study also evaluated the extracts' biofilm inhibition properties and monitored the expression of the lyt gene, integral to autolysis. The results prominently showed that the CRET70 extract demonstrated remarkable antibacterial strength. It achieved an MIC of 0.125 µg/mL against both tested S. pneumoniae strains. The disk diffusion assay recorded inhibition zones of 22.17 mm for ATCC 49619 and 17.20 mm for ATCC 70067. Impressively, CRET70 resulted in a 2-log decrease in bacterial numbers for both strains, showcasing its potent bactericidal capacity. The extract was also effective in inhibiting 77.40% of biofilm formation. Additionally, the significant overexpression of the lytA gene in the presence of CRET70 pointed to a potential mechanism of action for its antibacterial effects. The outcomes provided new perspectives on the use of Coptis rhizome in combating S. pneumoniae, especially significant in an era of escalating antibiotic resistance.

Onset and progression of postmortem histological changes in the central nervous system of RccHan™: WIST rats.

Death initiates a cascade of physiological and biochemical alterations in organs and tissues, resulting in microscopic changes that challenge the histopathological evaluation. Moreover, the brain is particularly susceptible to artifacts owing to its unique composition and its location within the cranial vault. The aim of this study was to compile and illustrate the microscopic changes in the central nervous system (CNS) of rats subjected to delayed postmortem fixation. It also scrutinizes the influence of exsanguination and cooling methods on the initiation and progression of these alterations. Twenty-four Wistar Han outbred rats (RccHan™: WIST) were sacrificed and stored either at room temperature (18-22°C) or under refrigeration (2-4°C). Necropsies were conducted at different time points postmortem (i.e., 0.5 h, 1 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 7 days and 14 days). Brain sections underwent simultaneous digital evaluation by 14 pathologists until a consensus was reached on terminology, key findings, and intensity levels. Microscopic observations varied among cell types. Glial cells were similarly affected throughout the CNS and showed pericellular halo, chromatin condensation and nuclear shrinkage. Neurons showed two types of postmortem changes as most of them showed progressive shrinkage, cytoplasmic dissolution and karyorrhexis whereas others acquired a dark-neuron-like appearance. Neuronal changes showed marked differences among neuroanatomical locations. Additional postmortem changes encompassed: granulation and microcavitation in neuropil and white matter; retraction spaces; detachment of ependyma, choroid plexus, and leptomeninges. Severity of findings after 48 h at room temperature was higher than after seven days under refrigeration and similar to or slightly lower than after 14 days under refrigeration. No clear differences were observed related to the sex or weight of the animals or their exsanguination status. This work elucidates the onset and progression of autolytic changes in the brains of Wistar Han rats, offering insights to accurately identify and enhance the histopathological evaluation.

An analysis combining proteomics and transcriptomics revealed a regulation target of sea cucumber autolysis.

Sea cucumber is a valuable seafood product and autolysis is the main concern for the aquaculture industry. This study employed proteomics and transcriptomics to investigate the autolysis mechanism of sea cucumbers. The fresh sea cucumber was exposed to UV light to induce autolysis. The body wall samples were cut off to analyze by proteomics and transcriptomics. The angiotensin-converting enzyme (ACE) inhibitor of teprotide and the activator of imatinib were gastric gavage to live sea cucumbers, respectively, to identify the regulation target. Autolysis occurrence was evaluated by appearance, soluble peptide, and hydroxyproline content. Four gene-protein pairs were ACE, AJAP10923, Heme-binding protein 2-like, and Ficolin-2-like. Only the ACE protein and gene changed synchronously and a significant down-regulation of ACE occurred in the autolysis sea cucumbers. Teprotide led to a 1.58-fold increase in the TCA-soluble protein content and a 1.57-fold increase in hydroxyproline content. No significant differences were observed between imatinib-treated sea cucumbers and fresh ones regarding TCA-soluble protein content or hydroxyproline levels (P > 0.05). ACE inhibitor accelerated the autolysis of sea cucumber, but ACE activator inhibited the autolysis. Therefore, ACE can serve as a regulatory target for autolysis in sea cucumbers.

Effect of humidity on postmortem changes in rats.

IMPORTANCE: In veterinary forensic science, accurately determining the postmortem interval (PMI) is crucial for identifying the causes of animal deaths. Autolysis, a significant postmortem process, influences PMI estimation, but its relationship with humidity is not well understood. OBJECTIVE: This study aimed to improve the accuracy of PMI estimates in veterinary forensic cases by looking into how different humidity levels affect autolysis in different organs of rats. METHODS: The study involved 38 male rats, examining histopathological changes in their heart, liver, and pancreas. These organs were subjected to controlled humidity levels (20%, 55%, and 80%) at a constant 22°C. Tissue samples were collected at several intervals (0 h, 12 h, 24 h, 3 days, and 8 days) for comprehensive analysis. RESULTS: Distinct autolytic characteristics in animal organs emerged under varying humidity conditions. The low-humidity environment rapidly activated autolysis more than the high-humidity environment. In addition, it was found that lower humidity caused nuclear pyknosis, cytoplasmic disintegration, and myofiber interruption. The liver, in particular, showed portal triad aggregation and hepatocyte individuation. The pancreas experienced cell fragmentation and an enlarged intracellular space. High humidity also caused the loss of striations in cardiac tissues, and the liver showed vacuolation. Under these conditions, the pancreas changed eosinophilic secretory granules. CONCLUSIONS AND RELEVANCE: The study successfully established a clear connection between the autolytic process in PMIs and relative humidity. These findings are significant for developing a more accurate and predictable method for PMI estimation in the field of veterinary forensic science.

New Insights into the Biological Functions of Essential TsaB/YeaZ Protein in Staphylococcus aureus.

TsaB/YeaZ represents a promising target for novel antibacterial agents due to its indispensable role in bacterial survival, high conservation within bacterial species, and absence of eukaryotic homologs. Previous studies have elucidated the role of the essential staphylococcal protein, TsaB/YeaZ, in binding DNA to mediate the transcription of the ilv-leu operon, responsible for encoding key enzymes involved in the biosynthesis of branched-chain amino acids-namely isoleucine, leucine, and valine (ILV). However, the regulation of ILV biosynthesis does not account for the essentiality of TsaB/YeaZ for bacterial growth. In this study, we investigated the impact of TsaB/YeaZ depletion on bacterial morphology and gene expression profiles using electron microscopy and deep transcriptomic analysis, respectively. Our results revealed significant alterations in bacterial size and surface smoothness upon TsaB/YeaZ depletion. Furthermore, we pinpointed specific genes and enriched biological pathways significantly affected by TsaB/YeaZ during the early and middle exponential phases and early stationary phases of growth. Crucially, our research uncovered a regulatory role for TsaB/YeaZ in bacterial autolysis. These discoveries offer fresh insights into the multifaceted biological functions of TsaB/YeaZ within S. aureus.

Analysis of microbial community evolution, autolysis phenomena, and energy metabolism pathways in Pholiota nameko endophytes.

Introduction: Pholiota nameko is a widely consumed edible fungus. This study focuses on two crucial developmental stages of Pholiota nameko, namely, mycelium and ascospores. The objectives of this research were to investigate changes in microbial diversity and community structure during the growth of Pholiota nameko and to analyze the adaptability of the dominant strains to their respective habitats through metabolic. Methods: Specifically, we conducted second-generation sequencing of the 16S rRNA gene (Illumina) on samples obtained from these stages. In addition, we isolated and characterized endophytes present in Pholiota nameko, focusing on examining the impact of dominant endophyte genera on autolysis. We also conducted a metabolic pathway analysis. Results and discussion: The results unveiled 578,414 valid sequences of Pholiota nameko endophytic fungi. At the phylum level, the dominant taxa were Basidiomycota, Ascomycota, Zoopagomycota, and Mucoromycota. At the genus level, the dominant taxa observed were Pholiota, Inocybe, Fusarium, and Hortiboletus. For endophytic bacteria, we obtained 458,475 valid sequences. The dominant phyla were Proteobacteria, TM6, Firmicutes, and Bacteroidetes, while the dominant genera were Edaphobacter, Xanthomonas, Burkholderia, and Pseudomonas. Moreover, we identified the isolated strains in Pholiota nameko using 16S rDNA, and most of them were found to belong to the genus Pseudomonas, with Pseudomonas putida being the most prevalent strain. The findings revealed that the Pseudomonas putida strain has the ability to slow down the breakdown of soluble proteins and partially suppress the metabolic processes that generate superoxide anion radicals in Pholiota nameko, thereby reducing autolysis. Additionally, our results demonstrated that molybdenum enzyme-mediated anaerobic oxidative phosphorylation reactions were the primary energy metabolism pathway in the Pseudomonas putida strain. This suggests that the molybdenum cofactor synthesis pathway might be the main mechanism through which Pholiota nameko adapts to its complex and diverse habitats.

Inhibiting Peptidoglycan Hydrolase Alleviates MRSA Pneumonia Through Autolysin-Mediated MDP-NOD2 Pathway.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a cause of staph infection that is difficult to treat because of resistance to some antibiotics. A recent study indicated that diarylurea ZJ-2 is a novel antibacterial agent against multi-drug resistant Enterococcus faecium. In this work, we refined the bactericidal mechanism of ZJ-2 as a peptidoglycan (PG) hydrolase by affecting AtlA-mediated PG homeostasis. Methods: A wild-type strain (WT) and a mutant strain (ΔatlA) were used to investigate the effects of ZJ-2 on the cell wall, PG, and autolysin regulatory system by antimicrobial susceptibility testing, hemolytic toxin assay, microanalysis, autolysis assay, qRT-PCR, ELISA and mouse model of pneumonia. Results: The results revealed that ZJ-2 down-regulated the expression of genes related to peptidoglycan hydrolase (PGH) (sprX, walR, atlA, and lytM), and reduced the levels of PG, muramyl dipeptide (MDP), cytokines, and hemolytic toxin, while ΔatlA interfered with the genes regulation and PG homeostasis. In the mouse MRSA pneumonia model, the same trend was observed in the nucleotide oligomerization domain protein 2 (NOD2) and relative proinflammatory factors. Conclusion: ZJ-2 may act as a novel inhibitor of PG hydrolyse, disrupting autolysin-mediated PG homeostasis, and reducing inflammation by down-regulating the MDP-NOD2 pathway.

Purification and partial physical-chemical characterization of a new bovine trypsin proteoform (zeta-trypsin).

Recent advancements in enzyme research have unveiled a new proteoform of bovine trypsin, expanding our understanding of this well-characterized enzyme. While generally similar to other trypsins, this novel proteoform comprises three polypeptide chains, marking a significant difference in activity, kinetic properties, and conformational stability. Compared with the already known bovine trypsin proteoforms, the results showed a lower: activity, kcat and kcat.KM-1 and protein 'foldedness' ratio for the new proteoform. Molecular autolysis, a common feature in trypsin and chymotrypsin, has been explored through comparative physical chemistry properties with other proteoforms. This new proteoform of trypsin not only enriches the existing enzyme repertoire but also promises to shed light on the intricate physiological pathway for enzyme inactivation. Our results suggest that the new trypsin proteoform is one of the likely final pathways for enzyme inactivation in a physiological environment. This discovery opens up new avenues for further research into the functional implications of this new trypsin proteoform.

Characterization of the major autolysin (AtlC) of Staphylococcus carnosus.

BACKGROUND: Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an accelerating effect, as described in many studies on cheese ripening. In contrast, very little is known about autolysis of starter cultures used in other fermentations. Staphylococcus (S.) carnosus is often used in raw sausage fermentations, contributing to nitrate reduction and flavor formation. In this study, we analyzed the influence of PGHs of the strains S. carnosus TMW 2.146 and S. carnosus TMW 2.2525 on their autolytic behavior. The staphylococcal major autolysin (Atl), a bifunctional enzyme with an N-acetylmuramoyl-L-alanine amidase and a glucosaminidase as an active site, is assumed to be the enzyme by which autolysis is mainly mediated. RESULTS: AtlC mutant strains showed impaired growth and almost no autolysis compared to their respective wild-type strains. Light microscopy and scanning electron microscopy showed that the mutants could no longer appropriately separate from each other during cell division, resulting in the formation of cell clusters. The surface of the mutants appeared rough with an irregular morphology compared to the smooth cell surfaces of the wild-types. Moreover, zymograms showed that eight lytic bands of S. carnosus, with a molecular mass between 140 and 35 kDa, are processed intermediates of AtlC. It was noticed that additional bands were found that had not been described in detail before and that the banding pattern changes over time. Some bands disappear entirely, while others become stronger or are newly formed. This suggests that AtlC is degraded into smaller fragments over time. A second knockout was generated for the gene encoding a N-acetylmuramoyl-L-alanine amidase domain-containing protein. Still, no phenotypic differences could be detected in this mutant compared to the wild-type, implying that the autolytic activity of S. carnosus is mediated by AtlC. CONCLUSIONS: In this study, two knockout mutants of S. carnosus were generated. The atlC mutant showed a significantly altered phenotype compared to the wild-type, revealing AtlC as a key factor in staphylococcal autolysis. Furthermore, we show that Atl is degraded into smaller fragments, which are still cell wall lytic active.

Preparation of Yeast Extract from Brewer's Yeast Waste and Its Potential Application as a Medium Constituent.

Yeast extract serves as a source of nutritional components essential for human dietary requirements, feed formulations, and the vital growth factors and nutrients necessary for microorganisms. However, the production cost of yeast extract using cultivated active dry yeast is relatively high. This study aims to utilize the autolysis of discarded yeast post beer brewing to produce yeast extract. The concentration, temperature, pH, and time conditions are systematically optimized. It reveals that the yield of amino nitrogen and solids in the extract was increased by 3.3% and 20.9% under the optimized conditions (1.2% wall-breaking enzyme, 1% yeast extract enzyme, and a hydrolysis time of 24 h) than that of the documented 4.03% and 69.05%. Additionally, a comparative analysis with commercially available yeast powder demonstrates that the yeast extract derived from this study adequately fulfills the nutritional requirements for microbial growth. Hence, the utilization of discarded beer yeast presents an opportunity for the valuable reclamation of waste yeast, showcasing promising potential applications.

Low-intensity ultrasound-assisted adaptive laboratory evolution of Bacillus velezensis for enhanced production of peptides.

This work aimed to explore low-intensity ultrasound-assisted adaptive laboratory evolution (US-ALE) of Bacillus velezensis and fermentation performance of mutant strains were investigated by nitrogen transformation metabolism. Results showed ultrasound accelerated the process of adaptive evolution and enhanced cell dry weight, amylase and protease activity of mutant strains, accompanied with the improved transformation abilities of NO-3-N to NH4+-N. Compared with original strain, the total peptide-N, peptide-N (<3 kDa) and autolytic peptide-N of mutant strains increased by the maximum 23.17%, 66.07% and 30.30%, respectively, based on ideal fermentation medium. According to the actual liquid-state fermentation of soybean meal and corn gluten meal with mutant strains, the highest peptide yields of 50.63% and 23.67% were noticed in mutant strain US-ALE-BV3, accompanied with the improved amino acid composition by bacterial autolysis technology. Thus, this study showed that low-intensity ultrasound could accelerate the process of adaptive evolution and US-ALE will provide more possibilities for modifying fermentation strains.

Perfusion Pressure and the Histology of Brain Death: A Unique Case in an Infant Maintained on Life Support.

Brain death is a not uncommon phenomena in the adult and pediatric population. Most cases are removed from life support soon after brain death is declared. Less commonly, systemic perfusion is maintained by life support for some time after neurologic function stops. These cases present uncommon opportunities to explore the histology of necrosis and autolysis in the context of global hypoxic ischemic damage. Here, we describe the unusual case of an infant maintained on life support for 2 weeks after brain death was declared with an emphasis on the resulting gross and histologic findings including a discussion of their underlying physiology.

Effects of autolysis and prolonged formalin fixation on histomorphology and immunohistochemistry of normal canine brain tissue: an experimental study.

CNS tumor diagnosis in dogs often relies on immunohistochemistry (IHC) given similar histologic features among tumors. Most CNS tissue samples encountered by diagnostic pathologists are collected during autopsy, and postmortem specimens can be susceptible to autolysis and prolonged formalin fixation, both of which have the potential to influence IHC results and interpretation. Here we evaluated the effects of experimentally controlled autolysis induced by delayed tissue fixation (sections of brain held for 2, 4, 8, 12, 24, 48, and 72 h in 0.9% NaCl at either room temperature or 37°C prior to fixation) as well as the effects of prolonged formalin fixation times (1 wk, 1 mo, 2 mo) on a panel of 8 IHC markers (CNPase, GFAP, Iba1, OLIG2, PGP9.5, MAP2, NeuN, synaptophysin) relevant to brain tumor diagnosis. Prolonged fixation of up to 2 mo had no detrimental effect on any immunomarker except NeuN, which had reduced immunolabeling intensity. Delayed fixation led to autolytic changes as expected, on a gradient of severity corresponding to increased time in saline prior to fixation. Several immunomarkers should be used with caution (CNPase, OLIG2) or avoided entirely (MAP2, NeuN) in markedly autolyzed brain and brain tumor tissues. Our results suggest that autolysis has minimal effect on most immunomarkers, but that advanced autolysis may cause a loss of specificity for GFAP, MAP2, and PGP9.5, a loss of intensity of CNPase and OLIG2, and loss of labeling with MAP2 and NeuN. Prolonged fixation affected only NeuN, with mildly decreased intensity.

Identification and investigation of the effects of N-acetylmuramoyl-L-alanine amidase in Bacillus amyloliquefaciens for the cell lysis and heterologous protein production.

Bacillus amyloliquefaciens (BA) is considered as an important industrial strain for heterologous proteins production. However, its severe autolytic behavior leads to reduce the industrial production capacity of the chassis cells. In this study, we aimed to evaluate the autolysis of N-acetylmuranyl-L-alanine amidase in BA TCCC11018, and further slowed down the cell lysis for improved the heterologous protein production by a series of modifications. Firstly, we identified six N-acetylmuramic acid-L-alanines by bioinformatics, and analyzed the transcriptional levels at different culture time points by transcriptome and quantitative real-time PCR. Then, by establishing an efficient CRISPR-nCas9 gene editing method, N-acetylmuramic acid-L-alanine genes were knocked out or overexpressed to verify its effect on cell lysis. Then, by single or tandem knockout N-acetylmuramic acid-L-alanines, it was determined that the reasonable modification of LytH and CwlC1 can slow down cell lysis. After 48 h of culture, the autolysis rate of the mutant strain BA ΔlytH-cwlC1 decreased by 4.83 %, and the amylase activity reached 176 U/mL, which was 76.04 % higher than that of the control strain BA Δupp. The results provide a reference for mining the functional characteristics of autolysin in Bacillus spp., and provide from this study reveal valuable insights delaying the cell lysis and increasing heterologous proteins production.

Influence of yeast growth conditions and proteolytic enzymes on the amino acid profiles of yeast hydrolysates: Implications for taste and nutrition.

This study investigated the effects of aerobic and anaerobic growth and proteolytic enzymes on the amino acid content of yeast hydrolysates in relation to taste and nutrition. Saccharomyces cerevisiae ATCC5574 was grown under fed-batch aerobic or batch anaerobic conditions. Intracellular glutamic acid (Glu) concentrations were 18-fold higher in aerobic yeast. Hydrolysis with papain and alkaline protease released more amino acids (AA) than simple autolysis or hydrolysis with bromelain, most significantly when applied to aerobic yeast (∼2-fold increase). Autolysates and bromelain hydrolysates from aerobic yeast had low levels of bitter and essential AAs, with high levels of umami Glu. Papain and alkaline protease hydrolysates of aerobic yeast had high levels of umami, bitter and essential AAs. Autolysates/hydrolysates from anaerobic yeast had moderate, high, and low levels of bitter, essential and umami AAs. Selection of both yeast growth conditions and hydrolysis enzyme can manipulate the free AA profile and yield of hydrolysates.

Mutation of mltG increases peptidoglycan fragment release, cell size, and antibiotic susceptibility in Neisseria gonorrhoeae.

IMPORTANCE: Neisseria gonorrhoeae is unusual in that the bacteria release larger amounts of cell wall material as they grow as compared to related bacteria, and the released cell wall fragments induce inflammation that leads to tissue damage in infected people. The study of MltG revealed the importance of this enzyme for controlling cell wall growth, cell wall fragment production, and bacterial cell size and suggests a role for MltG in a cell wall synthesis and degradation complex. The increased antibiotic sensitivities of mltG mutants suggest that an antimicrobial drug inhibiting MltG would be useful in combination therapy to restore the sensitivity of the bacteria to cell wall targeting antibiotics to which the bacteria are currently resistant.

Reducing Cellular Autolysis of Bacillus subtilis to Improve Keratinase Production.

Bacillus subtilis has been shown to be an excellent expression host for keratinases due to its powerful secretion system. However, cellular autolysis limits its production capacity. Here, we selected seven genes with significantly upregulated transcript levels from 15 genes associated with cellular autolysis as knockout targets by qRT-PCR and constructed a total of 127 strains to reduce cellular autolysis. Among them, the biomass of B. subtilis BSΔXLPC-ker deficient in xpf, lytC, pcfA, and cwlC increased by 57%. This was confirmed by cell staining, green fluorescent protein imaging, and extracellular nucleic acid leakage assay. Keratinase activity was increased by 1.46-fold in the 5 L fermenter. In addition, the activities of nattokinase and subtilisin E were also increased by 1.50-fold and 1.43-fold, respectively, in the modified chassis cells, which further confirms the generalizability of the strategy. Thus, reducing cellular autolysis to increase the ability of B. subtilis to produce subtilisins is promising.

Simple & better - Accelerated cheese ripening using a mesophilic starter based on a single strain with superior autolytic properties.

In the manufacture of rennet-coagulated cheese, autolysis is a rate-limiting step for ripening. Previously, a highly autolytic and thermotolerant Lactococcus lactis strain, RD07, was generated, which in preliminary laboratory cheese trials demonstrated great potential as a cheese ripening accelerant. RD07 is proteinase positive (Prt+) and capable of metabolizing citrate (Cit+). In this study, we obtained two derivatives of RD07: EC8 lacking the citrate plasmid, and EC2 lacking the proteinase plasmid. EC2 and EC8 retained the autolytic properties of RD07, and autolyzed 20 times faster than Flora Danica (FD) and SD96, where the latter is the parent of RD07. The three strains EC2, EC8 and RD07 were used in a ratio of 90:8:2, to create a simple starter termed ERC. ERC was less sensitive to cooking when cultured in milk and autolyzed well after entering the stationary phase upon facing sugar starvation. The ERC starter was benchmarked against FD and SD96 in laboratory cheese trials. The free amino acid content in cheese prepared using the ERC culture was 31 % and 34 % higher than in cheese prepared using FD and SD96, respectively. Overall, the ERC culture resulted in a more rapid release of free amino acids. A large-scale (5000 L) Gouda cheese trial at a Danish dairy demonstrated that the single strain ERC starter was comparable in performance to FD + an adjunct Lactobacillus helveticus culture. Furthermore, a large-scale Danbo cheese trial demonstrated that ERC could reduce the ripening period by 50 % for long-term ripened (25 weeks) cheese, resulting in better cheese.