Enzymes for production of whey protein hydrolysates and other value-added products.

Whey is a byproduct of dairy industries, the aqueous portion which separates from cheese during the coagulation of milk. It represents approximately 85-95% of milk's volume and retains much of its nutrients, including functional proteins and peptides, lipids, lactose, minerals, and vitamins. Due to its composition, mainly proteins and lactose, it can be considered a raw material for value-added products. Whey-derived products are often used to supplement food, as they have shown several physiological effects on the body. Whey protein hydrolysates are reported to have different activities, including antihypertensive, antioxidant, antithrombotic, opioid, antimicrobial, cytomodulatory, and immuno-modulatory. On the other hand, galactooligosaccharides obtained from lactose can be used as prebiotic for beneficial microorganisms for the human gastrointestinal tract. All these compounds can be obtained through physicochemical, microbial, or enzymatic treatments. Particularly, enzymatic processes have the advantage of being highly selective, more stable than chemical transformations, and less polluting, making that the global enzyme market grow at accelerated rates. The sources and different products associated with the most used enzymes are particularly highlighted in this review. Moreover, we discuss metagenomics as a tool to identify novel proteolytic enzymes, from both cultivable and uncultivable microorganisms, which are expected to have new interesting activities. Finally enzymes for the transformation of whey sugar are reviewed. In this sense, carbozymes with ß-galactosidase activity are capable of lactose hydrolysis, to obtain free monomers, and transgalactosylation for prebiotics production. KEY POINTS: • Whey can be used to obtain value-added products efficiently through enzymatic treatments • Proteases transform whey proteins into biopeptides with physiological activities • Lactose can be transformed into prebiotic compounds using ß-galactosidases.

Characterization of novel proteases identified by metagenomic analysis from dairy stabilization ponds.

Cheese whey is the main by-product of dairy industries. It is used as a raw material for other value-added products, like whey protein concentrate. By using enzymes, this product can be further treated to obtain new higher value products, like whey protein hydrolysates. Proteases (EC: 3.4) represent a large segment of industrial enzymes, since they are used in several industries, including food. In this work, we describe three novel enzymes identified using a metagenomic approach. Metagenomic DNA from dairy industry stabilization ponds were sequenced, and the predicted genes were compared against the MEROPS database, focusing on families commercially used to produce whey protein hydrolysates. From a total of 849 candidates, 10 were selected for cloning and expression and three showed activities with both the chromogenic substrate, azocasein, and whey proteins. Particularly, Pr05, an enzyme from the yet uncultured phylum Patescibacteria, showed activity that is comparable to a commercial protease. All these novel enzymes could represent an alternative for dairy industries to produce value-added products from industrial by-products. KEY POINTS: • Over 19,000 proteases were predicted in a sequence-based metagenomic analysis. • Three proteases were successfully expressed and showed activity with whey proteins. • The enzyme Pr05 showed hydrolysis profiles of interest for food industry.

Ruminal effects of excessive dietary sulphur in feedlot cattle.

Sulphur (S) dietary excess can limit productive performance and increase polioencephalomalacia (PEM) incidence in feedlot cattle (FC). Sulphur excess ingested is transformed to hydrogen sulphide (H2 S) by sulfo-reducing ruminal bacteria (SRB), being high ruminal H2 S concentration responsible for aforementioned damages. As the ruminal mechanisms involved in H2 S concentrations increase have not been elucidated, this study aimed to evaluate the ruminal environment, and the association between ruminal H2 S and dissimilatory SRB (DSRB) concentration in FC experimentally subjected to S dietary excess. Twelve crossbred steers were randomly assigned to one of two dietary S levels (6 animals per treatment): low (LS, 0.19% S) and high (HS, 0.39% S obtained by sodium sulfate inclusion at 0.86%). The study lasted 38 days, and on days 0, 22 and 38, ruminal gas samples were taken to quantify H2 S concentration, and ruminal fluid to determine total bacteria, DSRB, protozoa, volatile fatty acid and ammonia nitrogen concentration. For ruminal H2 S concentration, S dietary × sampling day interaction was significant (p < 0.001), so that the greater concentration was observed on days 22 and 38 with the HS diet. The remaining ruminal parameters were not affected by dietary S level, and no significant correlation between H2 S and DSRB concentrations was observed. The ruminal adaptation that maximizes H2 S production in FC consuming S excess does not seem to be associated with biological or biochemical alterations, nor DSRB concentration changes. The microbial diversity and ruminal environment were resilient to the S excess evaluated, suggesting that 0.39% of dietary S achieved by 0.86% sodium sulfate addition, could be used without disturbances on digestion nor health of FC.

Generalized glycogenosis in Brahman-derived breeds: diagnosis and prevalence in Argentina.

Generalized glycogenosis is a lethal autosomal recessive disease caused by a deficient activity of the acidic 1,4-α-glucosidase enzyme and characterized by an accumulation of glycogen within lysosomes. Three mutations in the GAA gene causing bovine generalized glycogenosis have been identified in two cattle breeds, Brahman and Shorthorn. The objective of this study was to evaluate the prevalence of carriers of the E7 mutation in the GAA gene in Argentinean Brahman-derived herds. A total of 930 Braford, 94 Brangus, and 8 Brahman samples were analyzed. The genotyping was done by polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP). We found that 12.02% (95% CI 12.00-12.04) of the total number of samples received were heterozygous (i.e., carriers) for the E7 mutation, while 12.58% (95% CI 12.56-12.60) of the Braford, 6.38% (95% CI 6.26-6.51) of the Brangus, and 12.50% (95% CI 9.82-15.18) of the Brahman samples were carriers of this loss-of-function allele. Neither breed nor sex were significantly associated to the presence of the mutation. The prevalence informed in this study is similar to the average prevalence reported for Australian Brahmans. The finding of heterozygous animals suggests that breeders and insemination centers should continue screening their herds to minimize the dissemination of this deleterious allele.

Omicron Waves in Argentina: Dynamics of SARS-CoV-2 Lineages BA.1, BA.2 and the Emerging BA.2.12.1 and BA.4/BA.5.

The COVID-19 pandemic has lately been driven by Omicron. This work aimed to study the dynamics of SARS-CoV-2 Omicron lineages during the third and fourth waves of COVID-19 in Argentina. Molecular surveillance was performed on 3431 samples from Argentina, between EW44/2021 and EW31/2022. Sequencing, phylogenetic and phylodynamic analyses were performed. A differential dynamic between the Omicron waves was found. The third wave was associated with lineage BA.1, characterized by a high number of cases, very fast displacement of Delta, doubling times of 3.3 days and a low level of lineage diversity and clustering. In contrast, the fourth wave was longer but associated with a lower number of cases, initially caused by BA.2, and later by BA.4/BA.5, with doubling times of about 10 days. Several BA.2 and BA.4/BA.5 sublineages and introductions were detected, although very few clusters with a constrained geographical distribution were observed, suggesting limited transmission chains. The differential dynamic could be due to waning immunity and an increase in population gatherings in the BA.1 wave, and a boosted population (for vaccination or recent prior immunity for BA.1 infection) in the wave caused by BA2/BA.4/BA.5, which may have limited the establishment of the new lineages.

Phylogenomic analysis for Campylobacter fetus ocurring in Argentina.

BACKGROUND AND AIM: Campylobacter fetus is one of the most important pathogens that severely affects livestock industry worldwide. C. fetus mediated bovine genital campylobacteriosis infection in cattle has been associated with significant economic losses in livestock production in the Pampas region, the most productive area of Argentina. The present study aimed to establish the genomic relationships between C. fetus strains, isolated from the Pampas region, at local and global levels. The study also explored the utility of multi-locus sequence typing (MLST) as a typing technique for C. fetus. MATERIALS AND METHODS: For pangenome and phylogenetic analysis, whole genome sequences for 34 C. fetus strains, isolated from cattle in Argentina were downloaded from GenBank. A local maximum likelihood (ML) tree was constructed and linked to a Microreact project. In silico analysis based on MLST was used to obtain information regarding sequence type (ST) for each strain. For global phylogenetic analysis, a core genome ML-tree was constructed using genomic dataset for 265 C. fetus strains, isolated from various sources obtained from 20 countries. RESULTS: The local core genome phylogenetic tree analysis described the presence of two major clusters (A and B) and one minor cluster (C). The occurrence of 82% of the strains in these three clusters suggested a clonal population structure for C. fetus. The MLST analysis for the local strains revealed that 31 strains were ST4 type and one strain was ST5 type. In addition, a new variant was identified that was assigned a novel ST, ST70. In the present case, ST4 was homogenously distributed across all the regions and clusters. The global analysis showed that most of the local strains clustered in the phylogenetic groups that comprised exclusively of the strains isolated from Argentina. Interestingly, three strains showed a close genetic relationship with bovine strains obtained from Uruguay and Brazil. The ST5 strain grouped in a distant cluster, with strains obtained from different sources from various geographic locations worldwide. Two local strains clustered in a phylogenetic group comprising intercontinental Campylobacter fetus venerealis strains. CONCLUSION: The results of the study suggested active movement of animals, probably due to economic trade between different regions of the country as well as with neighboring countries. MLST results were partially concordant with phylogenetic analysis. Thus, this method did not qualify as a reliable subtyping method to assess C. fetus diversity in Argentina. The present study provided a basic platform to conduct future research on C. fetus, both at local and international levels.

ß-Galactosidases from a Sequence-Based Metagenome: Cloning, Expression, Purification and Characterization.

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to its composition, mainly lactose and proteins, it can be considered as a raw material for value-added products, through physicochemical or enzymatic treatments. ß-Galactosidases (EC 3.2.1.23) are lactose modifying enzymes that can transform lactose in free monomers, glucose and galactose, or galactooligosacharides. Here, the identification of novel genes encoding ß-galactosidases, identified via whole-genome shotgun sequencing of the metagenome of dairy industries stabilization ponds is reported. The genes were selected based on the conservation of catalytic domains, comparing against the CAZy database, and focusing on families with ß-galactosidases activity (GH1, GH2 and GH42). A total of 394 candidate genes were found, all belonging to bacterial species. From these candidates, 12 were selected to be cloned and expressed. A total of six enzymes were expressed, and five cleaved efficiently ortho-nitrophenyl-ß-galactoside and lactose. The activity levels of one of these novel ß-galactosidase was higher than other enzymes reported from functional metagenomics screening and higher than the only enzyme reported from sequence-based metagenomics. A group of novel mesophilic ß-galactosidases from diary stabilization ponds' metagenomes was successfully identified, cloned and expressed. These novel enzymes provide alternatives for the production of value-added products from dairy industries' by-products.

Single nucleotide polymorphisms in candidate genes associated with milk yield in Argentinean Holstein and Holstein x Jersey cows.

BACKGROUND: Research on loci influencing milk production traits of dairy cattle is one of the main topics of investigation in livestock. Many genomic regions and polymorphisms associated with dairy production have been reported worldwide. In this context, the purpose of this study was to identify candidate loci associated with milk yield in Argentinean dairy cattle. A database of candidate genes and single nucleotide polymorphisms (SNPs) for milk production and composition was developed. Thirty-nine SNPs belonging to 22 candidate genes were genotyped on 1643 animals (Holstein and Holstein x Jersey). The genotypes obtained were subjected to association studies considering the whole population and discriminating the population by Holstein breed percentage. Phenotypic data consisted of milk production values recorded during the first lactation of 1156 Holstein and 462 Holstein x Jersey cows from 18 dairy farms located in the central dairy area of Argentina. From these records, 305-day cumulative milk production values were predicted. RESULTS: Eight SNPs (rs43375517, rs29004488, rs132812135, rs137651874, rs109191047, rs135164815, rs43706485, and rs41255693), located on six Bos taurus autosomes (BTA4, BTA6, BTA19, BTA20, BTA22, and BTA26), showed suggestive associations with 305-day cumulative milk production (under Benjamini-Hochberg procedure with a false discovery rate of 0.1). Two of those SNPs (rs43375517 and rs135164815) were significantly associated with milk production (Bonferroni adjusted p-values < 0.05) when considering the Holstein population. CONCLUSIONS: The results obtained are consistent with previously reported associations in other Holstein populations. Furthermore, the SNPs found to influence bovine milk production in this study may be used as possible candidate SNPs for marker-assisted selection programs in Argentinean dairy cattle.

Draft Genome Sequences of Escherichia coli O157:H7 Strains Rafaela_II (Clade 8) and 7.1_Anguil (Clade 6) from Cattle in Argentina.

Escherichia coli O157:H7 is a major etiologic agent of diseases in humans that cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. Here, we report the draft genome sequences of two strains isolated from cattle that had high levels of Shiga toxin 2 and high lethality in mice.