The Impact of the Adjunct Heat-Treated Starter Culture and Lb. helveticus LH-B01 on the Proteolysis and ACE Inhibitory Activity in Dutch-Type Cheese Model during Ripening

Adjunct cultures are used in cheesemaking to improve the sensory characteristics of the ripened cheeses. In addition, it is known that different adjunct cultures are capable of producing enzymes with the specificity to hydrolyze caseins, leading to the release of various bioactive compounds (bioactive peptides, amino acids, etc.). The objective of this study was to evaluate the effect of adjunct heat-treated starter XT–312 and a cheese culture Lb. helveticus LH-B01 on the proteolytic activity and angiotensin converting enzymes inhibitors (ACE) in cheese models during ripening. Seven different cheese models were evaluated for: proteolytic activity using the spectrophotometric method with ortho-phthaldialdehyde (OPA), soluble nitrogen (SN), trichloroacetic acid-soluble nitrogen (TCA-SN) phosphotungstic acid-soluble nitrogen (PTA-SN), total nitrogen (TN), pH, contents of water, fat, as well as for total bacteria count (TBC), count of Lactococcus genus bacteria, count of Lb. helveticus, and number of non-starter lactic acid bacteria (NSLAB). Presence of adjunct bacterial cultures both in the form of a cheese culture LH-B01 and heat-treated XT–312 starter promoted primary and secondary proteolysis, which resulted in acceleration of the ripening process. ACE inhibitory activity and proteolytic activity was the highest throughout of ripening for cheese model with LH-B01 culture. The cheese models with the adjunct heat-treated starter were characterized by lower TBC, NSLAB and lower count of Lactococcus genus bacteria during ripening, compared to control cheeses.

Physiological characterisation of Calabrian dairy yeasts and their possible use as adjunct cultures for cheese making

Seventeen samples of Calabrian ewe’s milk, ewe’s cheese (Pecorino del Poro) made with raw milk, goat’s milk, and goat’s cheese (Caprino d’Aspromonte) made with raw milk were used to obtain 124 yeast isolates. The most abundant species was Debaryomyces hansenii (61.3%), followed by Candida zeylanoides (32.3%) and Kluyveromyces marxianus (3.2%). The enzymatic profile of 25 selected yeast strains was determined. Lastly, they were studied for their interaction with eight dairy lactic acid bacteria – four coccal-shaped and four rod-shaped. The best strains may be used as adjunct cultures for cheese making.

Safety Evaluation, Biogenic Amine Formation, and Enzymatic Activity Profiles of Autochthonous Enterocin-Producing Greek Cheese Isolates of the Enterococcus faecium/durans Group

Autochthonous single (Ent+) or multiple (m-Ent+) enterocin-producing strains of dairy enterococci show promise for use as bioprotective adjunct cultures in traditional cheese technologies, provided they possess no pathogenic traits. This study evaluated safety, decarboxylase activity, and enzymatic (API ZYM) activity profiles of nine Ent+ or m-Ent+ Greek cheese isolates previously assigned to four distinct E. faecium (represented by the isolates KE64 (entA), GL31 (entA), KE82 (entA-entB-entP) and KE77 (entA-entB-entP-bac31)) and two E. durans (represented by the isolates KE100 (entP) and KE108 (entP-bac31-cyl)) strain genotypes. No strain was β-hemolytic or harbored vanA and vanB or the virulence genes agg, ace, espA, IS16, hyl, or gelE. All strains were of moderate to high sensitivity to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, penicillin, tetracycline, and vancomycin, except for the E. faecium KE64 and KE82 strains, which were resistant to erythromycin and penicillin. All cheese strains showed moderate to strong esterase-lipase and aminopeptidase activities and formed tyramine, but none formed histamine in vitro. In conclusion, all Ent+ or m-Ent+ strain genotypes of the E. faecium/durans group, except for the cyl-positive E. durans KE108, were safe for use as adjunct cultures in traditional Greek cheeses. Further in situ biotechnological evaluations of the strains in real cheese-making trials are required.

Lactic Acid Bacteria Adjunct Cultures Exert a Mitigation Effect against Spoilage Microbiota in Fresh Cheese

Lactic acid bacteria (LAB) have a strong mitigation potential as adjunct cultures to inhibit undesirable bacteria in fermented foods. In fresh cheese with low salt concentration, spoilage and pathogenic bacteria can affect the shelf life with smear on the surface and packaging blowing. In this work, we studied the spoilage microbiota of an Italian fresh cheese to find tailor-made protective cultures for its shelf life improvement. On 14-tested LAB, three of them, namely Lacticaseibacillus rhamnosus LRH05, Latilactobacillus sakei LSK04, and Carnobacterium maltaromaticum CNB06 were the most effective in inhibiting Gram-negative bacteria. These cultures were assessed by the cultivation-dependent and DNA metabarcoding approach using in vitro experiments and industrial trials. Soft cheese with and without adjunct cultures were prepared and stored at 8 and 14 °C until the end of the shelf life in modified atmosphere packaging. Data demonstrated that the use of adjunct cultures reduce and/or modulate the growth of spoilage microbiota at both temperatures. Particularly, during industrial experiments, C. maltaromaticum CNB06 and Lcb. rhamnosus RH05 lowered psychrotrophic bacteria of almost 3 Log CFU/g in a 5-week stored cheese. On the contrary, Llb. sakei LSK04 was able to colonize the cheese but it was not a good candidate for its inhibition capacity. The combined approach applied in this work allowed to evaluate the protective potential of LAB strains against Gram-negative communities.