Carlos Jiménez-Pérez

This study evaluated the effect of two concentrations oftrichloroacetic acid (TCA) on protein removal during theextraction of exopolysaccharides (EPS) produced by milk fermentationusing kefir grains (KGs) or Lactobacillus delbrueckiisubsp. bulgaricus NCFB 2772 (Lc2772). The results showedthat treatment with 10% TCA solution yielded more lyophilizedsolids (LS) in both fermentations compared to 80%TCA. Moreover, the solids obtained with 10% TCA exhibitedhigher sugar concentrations—34.95 ± 0.78 and 23.22 ± 0.25mgeq glucose mL-1 for LS-Lc2772 and LS-KGs, respectively. Incontrast, 80% TCA resulted in up to a 60% reduction in sugarcontent, suggesting potential polysaccharide hydrolysis.Although 80% TCA was more effective at removing proteins,it adversely affected EPS integrity, potentially compromisingtheir functional properties. These findings highlight the needto develop alternative protein removal methods that preservethe structural integrity of EPS.

This study evaluated the effect of sweet whey fermented by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772, an exopolysaccharide (EPS)-producing lactic acid bacterium (LAB), on the yield and quality of Requeson, a traditional Mexican fresh cheese, further supplemented with the probiotic strain Lactobacillus casei 431. Fermentation of the whey at 42°C for 48 hours resulted in the production of 1.126 ± 0.12 mg _eq dextran/mL of EPS. The incorporation of this EPS-rich whey led to a marked (∼230%) increase in cheese yield and significantly enhanced textural properties, including greater hardness, cohesiveness and elasticity. Furthermore, L. casei 431 maintained viability above 10 ⁷ cfu/g throughout 21 days of refrigerated storage (4°C), with no detectable microbial contamination. Although formulations containing EPS were slightly preferred in sensory evaluations, they exhibited improved stability and retained overall acceptable sensory quality. These results highlight the potential of EPS-producing LAB and probiotics to improve the technological, nutritional and functional attributes of fresh cheese, while contributing to the valorisation of dairy by-products.

BACKGROUND: Cow's milk is one of the most widely consumed foods worldwide due to its high nutritional value and rich content of biologically beneficial proteins. However, aflatoxin M1 (AFM1), a mycotoxin recognized for its carcinogenic potential, has been detected in milk, where it can bind to proteins. This study compared three methods for extracting AFM1 from commercial milk samples before and after enzymatic digestion with pepsin and pancreatin. RESULTS: Initial AFM1 concentrations and those following in vitro enzymatic digestion were measured using high‐performance liquid chromatography. Extraction method 3 proved the most effective, combining acetonitrile with ultrasound‐assisted extraction. AFM1 was detected in all milk samples at concentrations ranging from 0.25 to 12 μg L ⁻¹ , exceeding national (0.5 μg L ⁻¹ ) and international (0.05 μg L ⁻¹ ) regulatory limits. CONCLUSION: Enzymatic digestion increased AFM1 levels 3.66‐fold in four liquid milk samples, suggesting that raw milk measurements might underestimate consumer exposure. Thus, evaluating AFM1 bioaccessibility is essential for accurate toxicological risk assessment of milk and dairy products. © 2025 Society of Chemical Industry.

Aflatoxin M1 (AFM1), a toxic metabolite of aflatoxin B1 (AFB1), is commonly found in cow’s milk (CM) when dairy cattle consume contaminated feed. Chronic exposure to AFM1 poses serious health risks, particularly for children. This study aimed to quantify AFM1 levels in commercial CM available in Mexico and assess the associated carcinogenic risk across six age groups. A total of 23 milk samples were collected between March and July 2023 from supermarkets and convenience stores. AFM1 was measured using high-performance liquid chromatography (HPLC). Estimated daily intake (EDI), carcinogenic risk index (CRI), and Monte Carlo simulations (MCS) to model exposure were employed. AFM1 was detected in all samples, with 80% exceeding the legal limits set by Mexican and European regulations. The highest CRI values were observed in children aged 1–4 years (CRI > 5 × 10³), indicating an elevated risk. There were no statistically significant differences in AFM1 concentration among different milk or thermal treatments. A cluster analysis revealed patterns related to AFM1 content, price, and processing methods. The widespread detection of AFM1 raises a public health concern, especially for vulnerable populations. This underscores the need for targeted regulatory strategies and improved control of aflatoxins in the dairy production chain.

The cheese industry produces millions of tons of lactose-rich whey yearly, of which 50% is discharged into water and soil, leading to significant environmental challenges. In Mexico, cheese whey is repurposed for traditional cheese production. However, another by-product named secondary whey (SW) remains. This study focused on the production of exopolysaccharides (EPSs) via SW fermentation of kefir grains, yielding 632.6 ± 30.8 mg/L of a freeze-dried solid, and the carbohydrate-to-protein ratio stood at 2.89, corresponding to the bound EPS protein content. Through the analysis of Fourier Transform Infrared Spectroscopy (FTIR) spectra, it was determined that EPSs were successfully produced, as a signal was observed between 1200 and 1000 cm⁻¹, characteristic of the glycosidic bonds in polysaccharides.

Kefiran is an exopolysaccharide produced by milk fermentation that is widely used in the food industry, mainly to improve the rheological properties of foods, as well as for the formation of biofilms. Most of these properties are related to its interaction with milk proteins. It has been reported that kefiran has a molecular weight of up to 10⁷ Da and that it is formed by glucose and galactose molecules in equimolar quantities; however, its folding form is not completely understood. In this work it was elucidated that a model of the 20 kDa kefiran folds in a helical shape. In addition, it was determined that whey proteins can form complexes with kefiran.

Regular intake of fucosylated oligosaccharides has been associated with several benefits for human health, particularly for new-borns. Since these biologically active molecules can be found naturally in human milk, research efforts have been focused on the alternative synthetic routes leading to their production. In particular, utilization of fucosidases to perform stereoselective transglycosylation reactions has been widely investigated. Other reasons that bring these enzymes to the spotlight are their role in viral infections and cancer proliferation. Since their involvement in the pathogenesis of these diseases have been widely described, fucosidases have become a target in newly developed therapies. Finally, activity disorders of biologically important fucosidases can lead to health problems such as fucosidosis. What is common for both mechanisms is the interaction between the enzyme and substrates in and around the active site. Therefore, this review will analyse different substrate structures that have been tested in terms of their interaction with fucosidases active sites, either in synthesis or inhibition reactions. The published results will be compared from this perspective.

Human milk is the ideal food for newborns. In addition, thanks to the fucooligosaccharides it contains, it provides protection against gastrointestinal diseases. These oligosaccharides can be synthesized enzymatically using α-L-fucosidases. The aim of this work was to compare the active site of three fucosidases of the GH29 family with in silico methods: Thermotoga maritima, Lactobacillus casei, and Bifidobacterium longum subsp. infantis to understand the interaction occurring in the enzyme’s active site. In addition, the interaction with the substrate was also studied through molecular docking with pNP-fucose, a substrate used as a fucosyl donor to synthesize fucooligosaccharides. These in silico analyzes showed that the differences in the sequence between fucosidases of the GH29 family are associated with structural characteristics that intervene in the interaction of the substrate in the catalytic site, finding that the affinity of the T. maritima fucosidase and the pNP-fucose is the largest among the enzymes evaluated in this work, coinciding with that reported by in vitro methods.