Effect of enzymatic hydrolysis with Alcalase or Protamex on technological and antioxidant properties of whey protein hydrolysates.

The aim of this study was to evaluate the effect of the enzymatic hydrolysis, performed using Alcalase and Protamex enzymes, on the technological functionalities and the antioxidant capacity of whey protein hydrolysates (WPHs) to identify the conditions allowing to obtain target functionality/ies. Samples were characterized for hydrolysis degree (DH), molecular weight distribution, structural properties, and food-related functionalities. Free sulfhydryl groups and surface hydrophobicity significantly decreased with the increase in DH, regardless of the used enzyme. The foaming and antioxidant properties of Alcalase WPHs were higher as compared to those of WPI, reaching the maximum value at DH = 18-20 %, while higher DH resulted in impaired functionality. Gelling properties were guaranteed when WPI was hydrolysed by Protamex at DH < 15 % while foaming and antioxidant abilities were fostered at 15 < DH < 21 %. These results were well correlated with MW distribution and were rationalized into a road map which represents a useful tool in the selection of proper hydrolysis conditions (time, DH, enzyme type) to obtain WPHs with tailored functionalities. Research outcomes highlighted the possibility to drive protein hydrolysis to optimize the desired functionality/ies.

Unraveling the role of probiotics in affecting the structure of monoglyceride gelled emulsions: A low-field 1H NMR study.

The capacity of monoglyceride (MG) gelled emulsions (MEs) in protecting probiotic cells of Lacticaseibacillus rhamnosus against stresses suffered during food processing, storage, and human digestion has been recently demonstrated. These findings open new perspectives on the possible participation of probiotics in the stabilization of emulsion structure. To unravel this aspect, rheological analysis and Low-Field 1H NMR investigations were performed on MEs having different aqueous phases (water or skimmed milk) and stored for increasing time (1 and 14 days) at 4 °C. Loaded and unloaded samples were considered. Results highlighted that probiotics initially hindered the ability of MG to self-assemble in the multiphase environment, interacting in some way with MG crystalline lamellar structure, as confirmed by rheological and 1H NMR analysis. During storage, an increase of proton compartmentation was observed in loaded MEs indicating the role of probiotics in stabilizing MG structure at a molecular level. Such a result was more evident when the system was composed of milk, suggesting that the presence of milk-native components (i.e., lactose, proteins, and minerals) favored the cell-structure interactions. Such preliminary results could open new perspectives in considering probiotic cells as having an active role in the stabilization of food structure.

An integrated approach to explore the microbial biodiversity of natural milk cultures for cheesemaking.

The use of natural milk culture (NMC) represents a key factor in Protected Designation of Origin (PDO) Montasio cheese, contributing to its distinctive sensory profile. The complex microbial ecosystem of NMC is the result of heat treatment and incubation conditions, which can vary considerably among different production plants. In this study, the microbiota of NMC collected from 10 PDO Montasio cheese dairies was investigated by employing colony counts and metagenomic analysis. Furthermore, residual sugars, organic acids, and volatile profiles were quantitatively investigated. Results showed that Streptococcus thermophilus was the dominant species in all NMC, and a subdominant population made of other streptococci and Ligilactobacillus salivarius was also present. The incubation temperature appeared to be the main driver of biodiversity in NMC. Metagenomics allowed us to evidence the presence of minor species involving safety (e.g., Staphylococcus aureus) as well as possible functional aspects (Next Generation Probiotics). Statistical analysis based on residual sugars, organic acids, and volatiles' content allowed to correlate the presence of specific microbial groups with metabolites of great technological and sensory relevance, which can contribute to giving value to the artisanal production procedures of NMC and clarify their role in the creation of the characteristics of PDO Montasio cheese.

Volatilome of brine-related microorganisms in a curd-based medium.

The possible contribution of brine-derived microflora to the sensory attributes of cheese is still a rather unexplored field. In this study, 365 bacteria and 105 yeast strains isolated from 11 cheese brines were qualitatively tested for proteolytic and lipolytic activities, and positive strains were identified by sequencing. Among bacteria, Staphylococcus equorum was the most frequent, followed by Macrococcus caseolyticus and Corynebacterium flavescens. As for yeasts, Debaryomyces hansenii, Clavispora lusitaniae, and Torulaspora delbrueckii were most frequently identified. A total of 38% of bacteria and 59% of yeasts showed at least 1 of the metabolic activities tested, with lipolytic activity being the most widespread (81% of bacteria and 95% of yeasts). Subsequently 15 strains of bacteria and 10 yeasts were inoculated in a curd-based medium and assessed via headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry to determine their volatilome. After a 30-d incubation at 12°C, most strains showed a viability increase of about 2 log cfu/mL, suggesting good adaptability to the cheese environment. A total of 26 compounds were detected in the headspace, carbonyl compounds and alcohols being the major contributors to the volatile profile of the curd-based medium. Multivariate analysis was carried out to elucidate the overall differences in volatiles produced by selected strains. Principal component analysis and hierarchical clustering analysis demonstrated that the brine-related microorganisms were separated into 3 different groups, suggesting their different abilities to produce volatile compounds. Some of the selected strains have been shown to have interesting aromatic potential and to possibly contribute to the sensory properties of cheese.

Application of pre-adaptation strategies to improve the growth of probiotic lactobacilli under food-relevant stressful conditions.

While formulating a probiotic food, it is mandatory to make sure that the viability of probiotics is adequate at the point of consumption, which can be strongly compromised by stressful conditions due to low pH and high osmolarity. In this study, three probiotic lactobacilli were subjected to different pre-adaptation conditions, and the turbidimetric growth kinetics in challenging conditions (pH 4.0-6.5, NaCl 1-7%, sucrose 0.1-0.7 M) were evaluated. Different effects were observed for Lactobacillus acidophilus, Lacticaseibacillus casei, and Lactiplantibacillus plantarum. Indeed, pre-exposition to sub-optimal conditions in terms of pH and % NaCl significantly improved the ability of L. acidophilus and L. casei to overcome the osmotic stress due to salt or sucrose, and similar effects were observed for acidic stress. L. plantarum showed to be more tolerant to the challenging conditions applied in this study. Anyway, the pre-adaptation at conditions SUB_1 (pH 4.5 and NaCl 4%) and SUB_2 (pH 5 and NaCl 2%) speeded-up its growth kinetics by reducing the length of the lag phase under sucrose stress and enhancing the maximum growth rate at the highest pH tested. Moreover, an improvement in biomass amount was observed under sucrose stress. The whole data evidenced that the application of the appropriate pre-adaptation condition could contribute to making probiotics more robust towards challenging conditions due to food matrix, processing, and storage as well as gastrointestinal transit. Further studies will be necessary to gain insight into the proteomics and metabolomics responsible for increased tolerance to stressful conditions.

Raw milk preservation by hyperbaric storage: Effect on microbial counts, protein structure and technological functionality.

The possibility to apply hyperbaric storage (HS) at room temperature (20 °C) as a sustainable approach for preservation of raw skim milk was studied. Samples were stored at 200 and 150 MPa for up to 6 days. Optimal pressure for milk HS was found to be 150 MPa, since no clotting was detected for up to 6 days. 150 MPa-HS caused the irreversible inactivation of inoculated Escherichia coli (5.13 ± 0.33 logCFU mL-1) and Staphylococcus aureus (5.66 ± 0.93 logCFU mL-1) within 2 and 6 days, respectively. Inactivation of total and faecal coliforms (3.0 log reductions) below the detection limit was achieved after just 2 days, whereas lactic acid bacteria and coagulase-positive Staphylococci were inactivated after 6 days. Pressurized storage also caused an increase in proteose peptones and the release of submicelles from casein micelles. Micelles progressively aggregated with pressure-unfolded ß-Lactoglobulin. These phenomena led to milk presenting up to 4-fold better foaming capacity, probably due to ß-Lactoglobulin unfolding or higher proteose peptones content. This work demonstrated the capability of HS to guarantee milk preservation during storage, and brought attention on the opportunity to consider the technology for milk pasteurization and functionality improvement.

Turbidimetric definition of growth limits in probiotic Lactobacillus strains from the perspective of an adaptation strategy.

The application of an adaptation strategy for probiotics, which may improve their stress tolerance, requires the identification of the growth range for each parameter tested. In this study, 4 probiotics (Lactobacillus acidophilus, Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, and Lactiplantibacillus plantarum) were grown under different pH, NaCl, and sucrose concentrations at 25°C, 30°C, and 37°C. Turbidimetric growth curves were carried out and lag phase duration, maximum growth rate, and amplitude (i.e., the difference between initial and stationary phase optical density) were estimated. Moreover, cell morphology was observed, and cell length measured. The growth response, as well as the morphological changes, were quite different within the 4 species. The L. acidophilus was the most sensitive strain, whereas L. plantarum was shown to better tolerate a wide range of stressful conditions. Frequently, morphological changes occurred when the growth curve was delayed. Based on the results, ranges of environmental parameters are proposed that can be considered suboptimal for each strain, and therefore could be tested. The quantitative evaluation of the growth kinetics as well as the morphological observation of the cells can constitute useful support to the choice of the parameters to be used in an adaptation strategy, notwithstanding the need to verify the effect on viability both in model systems and in foods.

Correction: Effect of the formulation and structure of monoglyceride-based gels on the viability of probiotic Lactobacillus rhamnosus upon in vitro digestion.

Correction for 'Effect of the formulation and structure of monoglyceride-based gels on the viability of probiotic Lactobacillus rhamnosus upon in vitro digestion' by Sofia Melchior et al., Food Funct., 2021, DOI: 10.1039/D0FO01788D.

Effect of the formulation and structure of monoglyceride-based gels on the viability of probiotic Lactobacillus rhamnosus upon in vitro digestion.

This research was conducted to evaluate the potential use of saturated monoglyceride (MG)-based gels in the protection of probiotics upon in vitro digestion. For this purpose, a Lactobacillus rhamnosus strain was inoculated into binary and ternary systems, containing MGs, a water phase composed of an aqueous solution at controlled pH or UHT skimmed milk, and in ternary gels, sunflower oil. Gel structure characterization was initially performed just after preparation and after 14 days of storage at 4 °C by rheological, mechanical, thermal, and microscopy analyses. Afterwards, probiotic viability upon in vitro digestion was evaluated. The results highlighted that all freshly prepared samples showed good capability to protect L. rhamnosus with the exception of the binary system containing milk. However, the digestion of samples after 14 days of storage showed that the ternary system containing skimmed milk exhibited the best protection performance ensuring a L. rhamnosus viability of almost 106 CFU g-1 at the end of the gastrointestinal passage. Confocal microscopy results demonstrated that bacterial cells were located prevalently within the aqueous domain near the monoglycerides and protein aggregates. Under these conditions, they can simultaneously achieve physical protection and find nutrients to survive environmental stresses. These findings suggest that MG-based gels can be proposed as efficient carriers of probiotic bacteria not only during food processing and storage but also upon digestion.

Effect of different biopolymer-based structured systems on the survival of probiotic strains during storage and in vitro digestion.

BACKGROUND: This study aimed to evaluate the protective effect of different biopolymer systems on the viability of two probiotics (Lactobacillus rhamnosus and Streptococcus thermophilus) during storage and in vitro digestion. Methylcellulose (MC), sodium alginate (SA), and whey protein (WP)-based structures were designed and characterized in terms of pH, rheological properties, and visual appearance. RESULTS: The results highlighted that the WP-system ensured probiotic protection during both storage and in vitro digestion. This result was attributed to a combined effect of the physical barrier offered by the protein gel network and whey proteins as a nutrient for microbes. On the other hand, surprisingly, the viscous methylcellulose-based system was able to guarantee good microbial viability during storage. However, this was not confirmed during in vitro digestion. The opposite results were obtained for sodium alginate beads. CONCLUSION: The results suggest that the capacity of a polymeric structure to protect probiotic bacteria is a combination of structural organization and system formulation. © 2020 Society of Chemical Industry.

Metagenomic profiles of different types of Italian high-moisture Mozzarella cheese.

The microbiota of different types of Italian high-moisture Mozzarella cheese produced using cow or buffalo milk, acidified with natural or selected cultures, and sampled at the dairy or at the mass market, was evaluated using a Next Generation Sequencing approach, in order to identify possible drivers of the bacterial diversity. Cow Mozzarella and buffalo Mozzarella acidified with commercial cultures were dominated by Streptococcus thermophilus, while buffalo samples acidified with natural whey cultures showed similar prevalence of L. delbrueckii subsp. bulgaricus, L. helveticus and S. thermophilus. Moreover, several species of non-starter lactic acid bacteria were frequently detected. The diversity in cow Mozzarella microbiota was much higher than that of water buffalo samples. Cluster analysis clearly separated cow's cheeses from buffalo's ones, the former having a higher prevalence of psychrophilic taxa, and the latter of Lactobacillus and Streptococcus. A higher prevalence of psychrophilic species and potential spoilers was observed in samples collected at the mass retail, suggesting that longer exposures to cooling temperatures and longer production-to-consumption times could significantly affect microbiota diversity. Our results could help in detecting some kind of thermal abuse during the production or storage of mozzarella cheese.

Inactivation of Foodborne Bacteria Biofilms by Aqueous and Gaseous Ozone.

In this study, the efficacy of treatments with ozone in water and gaseous ozone against attached cells and microbial biofilms of three foodborne species, Pseudomonas fluorescens, Staphylococcus aureus, and Listeria monocytogenes, was investigated. Biofilms formed on AISI 304 stainless steel coupons from a mixture of three strains (one reference and two wild strains) of each microbial species were subjected to three types of treatment for increasing times: (i) ozonized water (0.5 ppm) by immersion in static condition, (ii) ozonized water under flow conditions, and (iii) gaseous ozone at different concentrations (0.1-20 ppm). The Excel add-in GinaFit tool allowed to estimate the survival curves of attached cells and microbial biofilms, highlighting that, regardless of the treatment, the antimicrobial effect occurred in the first minutes of treatment, while by increasing contact times probably the residual biofilm population acquired greater resistance to ozonation. Treatment with aqueous ozone under static conditions resulted in an estimated viability reduction of 1.61-2.14 Log CFU/cm2 after 20 min, while reduction values were higher (3.26-5.23 Log CFU/cm2) for biofilms treated in dynamic conditions. S. aureus was the most sensitive species to aqueous ozone under dynamic conditions. With regard to the use of gaseous ozone, at low concentrations (up to 0.2 ppm), estimated inactivations of 2.01-2.46 Log CFU/cm2 were obtained after 60 min, while at the highest concentrations a complete inactivation (<10 CFU/cm2) of the biofilms of L. monocytogenes and the reduction of 5.51 and 4.72 Log CFU/cm2 of P. fluorescens and S. aureus respectively after 60 and 20 min were achieved. Considering the results, ozone in water form might be used in daily sanitation protocols at the end of the day or during process downtime, while gaseous ozone might be used for the treatment of confined spaces for longer times (e.g., overnight) and in the absence of personnel, to allow an eco-friendly control of microbial biofilms and consequently reduce the risk of cross-contamination in the food industry.

Diversity within Italian Cheesemaking Brine-Associated Bacterial Communities Evidenced by Massive Parallel 16S rRNA Gene Tag Sequencing.

This study explored the bacterial diversity of brines used for cheesemaking in Italy, as well as their physicochemical characteristics. In this context, 19 brines used to salt soft, semi-hard, and hard Italian cheeses were collected in 14 commercial cheese plants and analyzed using a culture-independent amplicon sequencing approach in order to describe their bacterial microbiota. Large NaCl concentration variations were observed among the selected brines, with hard cheese brines exhibiting the highest values. Acidity values showed a great variability too, probably in relation to the brine use prior to sampling. Despite their high salt content, brine microbial loads ranged from 2.11 to 6.51 log CFU/mL for the total mesophilic count. Microbial community profiling assessed by 16S rRNA gene sequencing showed that these ecosystems were dominated by Firmicutes and Proteobacteria, followed by Actinobacteria and Bacteroidetes. Cheese type and brine salinity seem to be the main parameters accountable for brine microbial diversity. On the contrary, brine pH, acidity and protein concentration, correlated to cheese brine age, did not have any selective effect on the microbiota composition. Nine major genera were present in all analyzed brines, indicating that they might compose the core microbiome of cheese brines. Staphylococcus aureus was occasionally detected in brines using selective culture media. Interestingly, bacterial genera associated with a functional and technological use were frequently detected. Indeed Bifidobacteriaceae, which might be valuable probiotic candidates, and specific microbial genera such as Tetragenococcus, Corynebacterium and non-pathogenic Staphylococcus, which can contribute to sensorial properties of ripened cheeses, were widespread within brines.

Monitoring dry-curing of S. Daniele ham by magnetic resonance imaging.

S. Daniele hams were collected at different stages during dry-curing and submitted to magnetic resonance imaging (MRI) according to the acquisition Spin-Echo sequences T1 and T2. The intensity of the MR signals in the images of the Semimembranosus, Semitendinosus, Rectus femoris and Biceps femoris muscles of the hams was computed and expressed in grey levels. Muscles were also submitted to traditional analyses, including aw, soluble solids, sodium chloride, total and water soluble nitrogen. T1 and T2 MR signals well described the evolution of the phenomena occurring in the different muscles during dry-curing. MR signal acquired in T2 mode well correlated with traditional indicators in Semitendinosus, Rectus femoris and Biceps femoris muscles. Predictive models estimating the value of aw, moisture, salt content and proteolysis extent on the basis of the MR signal intensity were proposed.

Identification of the Enterobacteriaceae in Montasio cheese and assessment of their amino acid decarboxylase activity.

The aim of the study was to identify the species of Enterobacteriaceae present in Montasio cheese and to assess their potential to produce biogenic amines. Plate count methods and an Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) approach, combined with 16S rDNA sequencing, were used to investigate the Enterobacteriaceae community present during the cheesemaking and ripening of 6 batches of Montasio cheese. Additionally, the potential decarboxylation abilities of selected bacterial isolates were qualitatively and quantitatively assessed against tyrosine, histidine, ornithine and lysine. The most predominant species detected during cheese manufacturing and ripening were Enterobacter cloacae, Escherichia coli and Hafnia alvei. The non-limiting physico-chemical conditions (pH, NaCl% and a(w)) during ripening were probably the cause of the presence of detectable levels of Enterobacteriaceae up to 120 d of ripening. The HPLC test showed that cadaverine and putrescine were the amines produced in higher amounts by almost all isolates, indicating that the presence of these amines in cheese can be linked to the presence of high counts of Enterobacteriaceae. 44 isolates produced low amounts of histamine (<300 ppm), and four isolates produced more than 1000 ppm of this amine. Only 9 isolates, belonging to the species Citrobacter freundii, Esch. coli and Raoultella ornithinolytica, appeared to produce tyramine. These data provided new information regarding the decarboxylase activity of some Enterobacteriaceae species, including Pantoea agglomerans, Esch. fergusonii and R. ornithinolytica.

Identification of milk origin and process-induced changes in milk by stable isotope ratio mass spectrometry.

Stable isotope values were used to develop a new analytical approach enabling the simultaneous identification of milk samples either processed with different heating regimens or from different geographical origins. The samples consisted of raw, pasteurized (HTST), and ultrapasteurized (UHT) milk from different Italian origins. The approach consisted of the analysis of the isotope ratio of δ¹³C and δ¹5N for the milk samples and their fractions (fat, casein, and whey). The main finding of this work is that as the heat processing affects the composition of the milk fractions, changes in δ¹³C and δ¹5N were also observed. These changes were used as markers to develop pattern recognition maps based on principal component analysis and supervised classification models, such as linear discriminant analysis (LDA), multivariate regression (MLR), principal component regression (PCR), and partial least-squares (PLS). The results give proof of the concept that isotope ratio mass spectroscopy can discriminate simultaneously between milk samples according to their geographical origin and type of processing.

The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process.

A molecular biology method based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) was developed to detect Clostridium spp. in cheese samples suspected of late blowing. Strains of Clostridium spp. and different Lactic Acid Bacteria species, obtained from international collections, were used to determine the experimental conditions for the PCR amplification and DGGE differentiation. DNA extracted directly from cheeses with late blowing symptoms was subjected to PCR and DGGE analysis and traditional agar plating was performed for samples pasteurized and enriched overnight. Moreover, volatile fatty acids were determined for comparison purposes. The PCR-DGGE results were in agreement with the plating performed, and only samples presenting DGGE bands migrating at the same position as Clostridium spp. bands, showed the presence of Clostridium colonies on Reinforced Clostridial Medium plates. Butyric acid contents were high (>100 mg/kg) in the cases of positive DGGE results, underlining the suitability of the protocol for the study of cheese spoilage. The sensitivity of the method is estimated to be 10(4) CFU/g.