Bacteria are capable of colonizing industrial processing surfaces creating biofilms on them which may adversely affect the quality and safety of products. Traditional cleaning-in-place (CIP) treatments using caustic and nitric acid solutions have been known to exhibit variable efficiency in eliminating biofilm bacteria. Here, we introduce enzymes as an alternative to traditional CIP treatments and discuss their mechanism of action against bacterial biofilms in cheese manufacturing. In addition, we discuss research gaps namely thermal stability, substrate specificity and residual activity of enzymes that may play a vital role in the selection of enzymes with optimal effectiveness against multi species biofilms. The outcome of this mini review will aid in the development of a novel and sustainable enzyme-based CIP treatment during cheese manufacturing in the future.

Making cheese manufacturing environmentally sustainable is a major concern in the integrated management of this industrial sector. This concern is mainly due to the environmental impact of the discharge of its wastewaters, carrying heavy loads of salinity, nutrients, organic matter, solids and oils and fats. These discharges must meet increasingly stringent quality requirements. Some physicochemical (e.g. coagulation-flocculation, precipitation, oxidation) and biological (e.g. aerobic and anaerobic bioreactors and wetlands) treatments have been studied to address this problem. However, these treatments involve costs that some producers cannot sustain, face difficulties with biological reactor operational stability and often fail to consistently produce effluents compatible with discharge standards. In this context, aiming at the design of a simple and economical treatment method, several precipitation processes were tested using a fixed dosage of CaCO3 (75 g/L), combined with different dosages of FeCl3, FeSO4 or Ca(OH)2. The goal of the treatment was to produce a supernatant that would be evaluated as to its suitability for discharge into natural water courses or municipal treatment systems, or for reuse applications. The generated sludge would be evaluated for possible agricultural valorization. Through the measurement of the relevant supernatant quality parameters and using statistical analysis, it was possible to choose the best dosages for each of the tested coagulants (1.0, 1.0 and 0.6 g/L for FeCl3, FeSO4 and Ca(OH)2, respectively). Among these, the most efficient treatment was obtained with CaCO3 75 g/L + FeSO4 1.0 g/L. For this best-case scenario, the treatment led to removal yield values of 55.1% for chemical oxygen demand (COD), 92.0% for total phosphorus, 95.7% for turbidity, 59.1% for total phenols, 94.3% for nitrates, 71.0% for nitrites, 51.0% for total solids (TS) and 97.2% for oils and fats. The treatment did not produce an effluent supernatant with adequate quality for direct discharge into water courses, serving however as an efficient pretreatment for agricultural reuse. All the sludges generated in these treatments showed good potential for agricultural valorization due to their high nutrient content, along with pH and conductivity values within the acceptable ranges for soil application. Thus, this work contributes for a better integration of the cheese manufacturing industry in the overall aims of water and nutrient resources recovery in rural, agricultural areas.

Cheese consumption has been gradually increased in China. However, both the manufacturing process of cheese and the utilization of its main by-product were not well developed. Based on the sensory evaluation, Box-Behnken Design (BBD) was performed in the present study to optimize the cheese processing, which was proved more suitable for Chinese. The optimal parameters were: rennet 0.052 g/L, start culture 0.025 g/L and CaCl2 0.1 g/L. The composition analysis of fresh bovine milk and whey showed that whey contained most of the soluble nutrients of milk, which indicated that whey was a potential resource of cyclic adenosine-3\', 5\'-monophosphate (cAMP). Thus, the cAMP was isolated from whey, the results of high-performance liquid chromatography (HPLC) analysis showed that the macroporous adsorption resins (MAR) D290 could increase the concentration of cAMP from 0.058 µmol/mL to 0.095 µmol/mL. We firstly purified the cAMP from the whey, which could become a new source of cAMP.

Bovine and camel chymosins are aspartic proteases that are used in dairy food manufacturing. Both enzymes catalyze proteolysis of a milk protein, κ-casein, which helps to initiate milk coagulation. Surprisingly, camel chymosin shows a 70% higher clotting activity than bovine chymosin for bovine milk, while exhibiting only 20% of the unspecific proteolytic activity. By contrast, bovine chymosin is a poor coagulant for camel milk. Although both enzymes are marketed commercially, the disparity in their catalytic activity is not yet well understood at a molecular level, due in part to a lack of atomistic resolution data about the chymosin-κ-casein complexes. Here, we report computational alanine scanning calculations of all four chymosin-κ-casein complexes, allowing us to elucidate the influence that individual residues have on binding thermodynamics. Of the 12 sequence differences in the binding sites of bovine and camel chymosin, eight are shown to be particularly important for understanding differences in the binding thermodynamics (Asp112Glu, Lys221Val, Gln242Arg, Gln278Lys. Glu290Asp, His292Asn, Gln294Glu, and Lys295Leu. Residue in bovine chymosin written first). The relative binding free energies of single-point mutants of chymosin are calculated using the molecular mechanics three dimensional reference interaction site model (MM-3DRISM). Visualization of the solvent density functions calculated by 3DRISM reveals the difference in solvation of the binding sites of chymosin mutants.

MicroRNAs (miRNAs) are abundant in bovine milk and milk derived from other livestock, and they have functional roles in infants and in the secretion process of mammary glands. However, few studies have evaluated miRNAs in dairy processes, such as during cheese making and ripening. Thus, we investigated the characteristics of milk-derived miRNAs during the manufacturing and ripening of Camembert cheese as well as the microbiota present using the quantitative reverse transcription polymer chain reaction (RT-qPCR) and 16S rRNA pyrosequencing, respectively. Pyrosequencing showed that the cheese microbiota changed dramatically during cheese processing, including during the pasteurization, starter culture, and ripening stages. Our results indicated that the RNA contents per 200 mg/200 μl of the sample increased significantly during cheese-making and ripening. The inner cheese fractions had higher RNA contents than the surfaces after 12 and 22 days of ripening in a timedependent manner (21.9 and 13.2 times higher in the inner and surface fractions than raw milk, respectively). We performed a comparative analysis of the miRNAs in each fraction by RT-qPCR. Large amounts of miRNAs (miR-93, miR-106a, miR-130, miR-155, miR-181a, and miR- 223) correlated with immune responses and mammary glands were present in aged cheese, with the exception of miR-223, which was not present on the surface. Considerable amounts of miRNAs were also detected in whey, which is usually disposed of during the cheese-making process. Unexpectedly, there were no significant correlations between immune-related miRNAs and the microbial populations during cheese processing. Taken together, these results show that various functional miRNAs are present in cheese during its manufacture and that they are dramatically increased in amount in ripened Camembert cheese, with differences according to depth.