Partial purification of linoleic acid isomerase enzyme from Lactobacillus paracasei bacteria isolated from milk / Purificação parcial da enzima isomerase do ácido linoleico da bactéria Lactobacillus paracasei isolada do leite

Conjugated Linoleic Acid (CLA) has attracted the attention of many researchers, especially that of microbial origin due to its biological importance to the consumer. The current study aims to extract LA Isomerase enzyme from Lactobacillus paracasei bacteria from milk and to use the enzyme in the production of CLA. Selective media, including MRS and MRS-Dagatose, were used in isolating local strains. The selected bacterial isolates were tested for their ability to produce LA-Isomerase enzyme. The isolate with high enzymatic activity was selected. After extraction and partial purification of the enzyme, the optimal conditions for the production of conjugated fatty acid were studied, and the reaction products were diagnosed using GC-MS technology. It was found that 11 isolates have the ability to produce CLA at different concentrations, H1 isolate showed the highest production of conjugated fatty acid at a concentration of 120.45 g.ml-1, this isolate was selected as the source for enzyme extraction. The enzymatic activity of the crude extract and partially purified with ammonium sulfate was estimated using color methods at wavelength of 233 nm. The effect of the optimum conditions (pH, temperature, linoleic acid concentration and enzyme concentration) on the CLA product was studied using the partially purified LA Isomerase enzyme, the optimum conditions for production were 6.5, 45 °C, 100 µg.ml-1 and 0.7 ml, respectively. The GC-MS technique showed the presence of a number of reaction products that are isomers of conjugated linoleic acid (C9T11, T9T12, T10C12) with different concentrations.

O Ácido Linoleico Conjugado (CLA) tem chamado a atenção de diversos pesquisadores, principalmente aquele de origem microbiana, devido à sua importância biológica para o consumidor. O presente estudo visa extrair a enzima LA Isomerase da bactéria Lactobacillus paracasei do leite e usar essa enzima na produção de CLA. Meios seletivos, incluindo MRS e MRS-Dagatose, foram usados no isolamento de cepas locais. Os isolados bacterianos selecionados foram testados quanto à sua capacidade de produzir a enzima LA-Isomerase. Foi selecionado o isolado com alta atividade enzimática. Após a extração e purificação parcial da enzima, as condições ideais para a produção de ácido graxo conjugado foram estudadas e os produtos da reação foram identificados usando a tecnologia GC-MS. Verificou-se que 11 isolados possuem capacidade de produzir CLA em diferentes concentrações. O isolado H1 apresentou a maior produção de ácido graxo conjugado, na concentração de 120,45 g.ml-1, e este isolado foi selecionado como fonte para extração enzimática. A atividade enzimática do extrato bruto e parcialmente purificado com sulfato de amônio foi estimada por métodos de coloração em comprimento de onda de 233 nm. O efeito das condições ótimas (pH, temperatura, concentração de ácido linoleico e concentração de enzima) no produto CLA foi estudado usando a enzima LA Isomerase parcialmente purificada e as condições ótimas para produção foram 6,5, 45 °C, 100 µg.ml-1 e 0,7 mL, respectivamente. A técnica de GC-MS mostrou a presença de uma série de produtos de reação que são isômeros do ácido linoleico conjugado (C9T11, T9T12, T10C12) com diferentes concentrações.

Ultrasensitive detection of glucose oxidase and alkaline phosphatase in milk based on valence regulated upconversion nanoprobes.

Fresh milk should undergo sterilization before consumption to eliminate bacteria that can cause foodborne illnesses. Additional antimicrobial measures are beneficial to extend its shelf life. The nanoprobe developed herein can not only inspect the activity of alkaline phosphatase (ALP) for evaluating the degree of pasteurization, but also detect the activity of glucose oxidase (GOD), which is added as a chemical preservative. The facile preparation of the nanoprobe involved introducing gallic acid-Fe complex (GA-Fe) into lanthanide doped upconversion nanomaterials (UCNPs). Based on the alteration of iron's valence state in the complex through a straightforward redox reaction, both enzyme activities could be determined through colorimetric and luminometric dual-signal readouts. With detection limits of 1.669 × 10-5 for GOD and 9.81 × 10-6 U/mL for ALP respectively, this nanoprobe shows merits of easy operation and high sensitivity. Successful application in milk samples demonstrates its potential as an innovative and cost-effective approach to food safety inspection.

Sensitive detection of aflatoxin B1 in foods by aptasensing-based qPCR.

In this study, a reproductive switch DNA template was designed using aptasensing principles for the accurate quantification of aflatoxins. The template transformed the aflatoxin molecule into linear DNA of 102 nt. The linear DNA was subjected to a quantitative polymerase chain reaction (qPCR) to determine its initial copy number, which was positively correlated with the aflatoxin concentration. Using aflatoxin B1 (AFB1) as a model, the established method could quantify AFB1 within the range of 10-16-10-11 Mol/mL (detection limit equals 0.03 pg/mL), with a linear correlation coefficient R2 of 0.974. Good anti-interference abilities against common food ingredients and high specificity towards other mycotoxins were demonstrated. The established method was successfully applied for the quantification of AFB1 in complex foods such as soy sauce, milk, yellow wine, and peanut butter. The design of a reproductive switch template introduces a novel approach for the sensitive detection of small-molecule toxicants in foods.

Gastric clot formation and digestion of milk proteins in static in vitro infant gastric digestion models representing different ages.

Gastric digestion conditions change during infancy from newborn towards more adult digestion conditions, which can change gastric digestion kinetics. However, how these changes in gastric digestion conditions during infancy affect milk protein digestion has not been investigated. Therefore, we aimed to investigate milk protein digestion with static in vitro gastric digestion models representing one-, three- and six-month-old infants. With increasing age, gastric clots and soluble proteins were digested more extensively, which may partly be attributed to the looser gastric clot structure. Larger differences with increasing age were found for heated than unheated milk proteins, which might be caused by the presence of denatured whey proteins. Taken together, these findings show that gastric milk protein digestion increases during infancy. These in vitro gastric digestion models could be used to study how milk protein digestion changes with infant age, which may aid in developing infant formulas for different age stages.

Aroma characteristics of flaxseed milk via GC-MS-O and odor activity value calculation: Imparts and selection of different flaxseed varieties.

Currently, the effect of varieties on the flavor and stability of flaxseed milk remains unknown. Therefore, this study was conducted to investigate the effects of different varieties on the stability, sensory, and aromas of flaxseed milk. 51 volatile compounds were identified in flaxseed milk using Stir Bar Sorptive Extraction-Gas Chromatography-Olfactometry-Mass Spectrometry (SBSE-GC-O-MS). Among them, 1-octen-3-ol, 2-methoxy-4-vinylphenol, and 2,3,5-trimethylpyrazine contributed higher relative odor active values (ROAV), resulted in the fruity, roasted, sweet, and cucumber in flaxseed milk. Isovaleraldehyde (green notes) was not detected in XHZM. However, other compounds such as 1-nonanol (floral), γ-nonanolactone and γ-octanoic lactone (coconut milk) had higher concentrations, causing a better sensory evaluation. Additionally, its stability was relatively good. The orthogonal partial least-squares regression (OPLS) model and VIP values showed that eight compounds were responsible for the sensory differences from different varietals. The study provided references to selection and understanding flavor composition basis of flaxseed milk.

Short-communication: Study of fatty acid metabolites in microbial conjugated fatty acids-enrichment of milk and discovery of additional undescribed conjugated linolenic acid isomers.

Microbially enriched food in conjugated linoleic (CLA) and conjugated linolenic (CLNA) acids is intensively studied nowadays. The conversion of linoleic (LA) and α-linolenic acids (α-LNA) into these compounds may involve different fatty acid (FA) intermediates. This research aimed to investigate potential FA byproducts in milk during microbial CLA/CLNA-enrichment using Bifidobacterium breve DSM 20091. Milk fermented with pure α-LNA showed a decrease in free myristic acid, while pure LA led to an increase in free stearic acid. No additional FA compounds were found alongside CLA/CLNA isomers. The strain produced several CLA isomers from LA, but only when administered alone. Nonetheless, when α-LNA was assayed, additional CLNA isomers, never reported before for bifidobacteria, were observed. In conclusion, except for stearic acid in the presence of LA, no side-FA metabolites were released during milk microbial CLA/CLNA-enrichment. Results suggest either CLA/CLNA production occurs in one single-step or intermediates biotransformation is very fast.

Sonocatalytic degrading antibiotics over activated carbon in cow milk.

Herein, an efficient, simple and economical approach to remove antibiotics (ABX), i.e. ceftiofur hydrochloride, sulfamonomethoxine sodium (SMM), marbofloxacin and oxytetracycline by sonication with activated carbon (AC) from cow milk has been successfully implemented. The pseudo-first-order kinetics constants for the sonolytic and sonocatalytic degrading SMM are 0.036 and 0.093 min-1, respectively. The synergistic efficiency of removing ABX by using sonocatalysis reached 1.8-4.0. Hydrophobic ABX underwent faster degradation than hydrophilic ABX in sonocatalytic systems. Adding 0.5 mmol L-1 H2O2, the optimal concentration, improved the sonocatalytic degradation rates of ABX by 9.1%-28.5%. Surface area and dose of AC play crucial roles in the sonocatalysis of ABX. By sonicating 50 mL of 5.52 µmol L-1 ABX in milk at 500 kHz and 259 W with 20 mg AC for 20-60 min resulted in residual ABX concentrations ranging from 42.6 to 95.1 µg L-1, which meet the relative maximum residue limits set by European Commission.

Microfluidic chip and chiroptical gold nanoparticle-based colorimetric sensor for enantioselective detection of L-tryptophan.

Herein, we introduce a novel integrated system that merges an enantio-discriminative bio-MOF-packed centrifugal microfluidic chip made from PDMS with a user-friendly on-site colorimetric sensor. This innovative approach enables the precise enantioselective recognition of L-tryptophane (L-Trp). This chiral recognition probe was successfully synthesized through meticulous control of nano-ovals-shaped gold nanoparticles morphology and surface passivation. The operational factor of this methodology was optimized to ensure simplicity, practicality, and efficiency. This optimization led to reduced reagent consumption and instantaneous analytical feedback. The integrated system was effectively applied for enantioselective separation and quantification of L-Trp across an extensive linear range of 50 µM-1.5 mM, impressive limit of detection as low as 15 µM. It is noteworthy that this integrated system demonstrated desirable selectivity even in the presence of similar biomolecules, showcasing its robust performance and rapid detection capability. Further extended the application of this strategy to exceptional performance across enantioselective sensing of L-Trp in various sample matrices, comprising bovine serum albumin, bovine milk, blood plasma and urine samples. This integrated microfluidic sample pretreatment, chiroptical sensing, and on-site signal recording with a smartphone hold tremendous potential for widespread implementation, practical applications engaging healthcare and environmental, food safety, and point-of-needs analysis, facilitating successive solution mixing and colorimetric detection.

Insight into differences in whey proteome from human and eight dairy animal species for formula humanization.

Human breastmilk fulfills the nutritional needs of infants and therefore is the best template for formula. In this study, whey proteins were investigated among human and eight dairy animal species using label-free proteomics approach. Totally, 965 proteins from milk whey were identified and large variations were observed between human and animals. Several proteins, including ß-galactosidase, fatty acid synthase, osteopontin, lactoferrin, mannose receptor, and complement C4-A, which are associated with digestion and immune response, exhibited significantly higher levels in human milk whey. Conversely, specific animal milk whey demonstrated elevated abundance of lipocalin 2, lysozyme, and glycosylation-dependent cell adhesion molecule 1. These differential proteins are enriched in complement and coagulation cascades, lysosome, and phagosome pathways. The findings shed light on the variations in the whey proteome composition between human and animal milk, which can contribute to optimizing formula humanization.

An electrochemiluminescence sensor based on the CNTs and CdSe@ZnSe for determination of melamine in milk samples.

In this work, a novel electrochemiluminescence (ECL) sensor based on the CdSe@ZnSe and CNTs was constructed for the detection of melamine. CdSe@ZnSe acted as the co-reaction promoter for the enhancement of Ru(bpy)32+/tri-n-propylamine (TPrA) system and CNTs acted as carriers to immobilize more CdSe@ZnSe. The initial ECL signal significantly amplified due to the synergistic effect of CNTs and CdSe@ZnSe. The ECL signal decreased with the addition of melamine, and the change value of ECL intensity (ΔI) was linearly related to the logarithm of melamine concentration. The constructed ECL sensor was able to detect melamine in the range of 1.0 × 10-11 - 1.0 × 10-7 M, and the detection limit was 3.3 × 10-12 M (S/N = 3). It can be used to detect melamine in milk samples.

Two birds with one stone: A universal design and application of signal-on labeled fluorescent/electrochemical dual-signal mode biosensor for the detection of tetracycline residues in tap water, milk and chicken.

An ingenious and universal design of fluorescent/electrochemical dual-signal mode sensing platform was constructed for the sensitive, selective and accurate detection of tetracycline (TET). Apt-functionalized nano-magnetic beads (Fe3O4-Apt) as capture probe, Apt-complementary short-chain functionalized fluorescent MOF loaded with methylene blue (MB) (cDNA-MOF-MB) as dual-signal tag were prepared. The sensing platform (Fe3O4-Apt/cDNA-MOF-MB) was formed based on the base complementary pairing of Apt and cDNA. With the help of Apt for target recognition, together with magnetic separation technology, a dual-signal mode biosensor was constructed. The dual-signal mode biosensor exhibited a wide linear concentration range from 1.00 × 10-9 g/mL to 1.00 × 10-4 g/mL with a low limit of detection (LOD) of 1.69 × 10-10 g/mL (fluorescence mode assay) and 1.15 × 10-10 g/mL (electrochemical mode assay). The proposed biosensor had been successfully applied to the determination of TET content in real samples with satisfactory recoveries (94.99-101.30%).

Co-fermented milk beverage has better stability and contains more health-promoting amino acid metabolites than single-strain-fermented milk beverage over one-month storage.

Few studies investigated the effects of co-fermentation with bifidobacteria on post-storage changes of probiotic fermented beverages (PFBs). Thus, this study compared the post-storage changes in physicochemical index and metabolomes of PFBs produced singly by Lacticaseibacillus paracasei PC-01 (PC-01) or in combination with Bifidobacterium adolescentis B8589 (B8589). No significant differences were observed in the pH, titratable acidity, and viable cell counts between the two PFBs over 30-day storage. However, adding B8589 not only increased the stability of PFB (based on evaluating differences in PFBs metabolomics), but also the contents of beneficial amino acid metabolites, including 4-hydroxystyrene, gamma-aminobutyric acid, N-acetyl-l-aspartic acid, d-alanyl-d-alanine, and l-malic acid, after storage. Our study showed that B8589 is preferred to single-strain fermentation by PC-01. This study supports the concept of using bifidobacteria as starter culture in PFB production.

Coconut milk treated by atmospheric cold plasma: Effect on quality and stability.

Commercial sterilization plays an important role in extending the shelf-life of coconut milk. However, thermal sterilization affects the quality of coconut milk. This study was initiated to evaluate the effects of atmospheric cold plasma (ACP) treatment on some important quality parameters of coconut milk. ACP treatment had a slight effect on physicochemical characteristics and nutritional ingredients while it obviously reduced the colony count. Furthermore, ACP treatment obviously promoted the formation of lactone, an indispensable volatile substance in coconut milk. Insufficient or moderate ACP treatment had subtle effect on the sensory quality. Notably, moderate ACP treatment reduced the droplet size from 28.0 µm to 18.6 µm, and improved the stability during storage and centrifugation, especially at 60 kV 60 s. Overall, sterilization of coconut milk by ACP at 60 kV 60 s was the most ideal. This study can provide theoretical guidance for the application of ACP in liquid food.

Detection mechanism and the outlook of metal-organic frameworks for the detection of hazardous substances in milk.

Milk has a high nutritional value. However, milk is easily contaminated in the production, processing, and storage processes, which harms consumers' health. Therefore, the harmful substances' detection in milk is important. Metal-organic frameworks (MOFs) have proven high potential in food safety detection due to their unique porous structure, large effective surface area, large porosity, and structural tunability. This article systematically describes the detection mechanism of fluorescence, electrochemical, colorimetric, and enzyme-linked immunosorbent assay based on MOFs. The progress of the application of MOFs in the detection of antibiotics, harmful microorganisms and their toxins, harmful ions, and other harmful substances in milk in recent years is reviewed. The structural tunability of MOFs enables them to be functionalized, giving the ability to be applied to different detection methods or substances. Therefore, MOFs can be used as an advantageous sensing material for detecting harmful substances in the complex environment of milk.

Electrochemical sensor for sensitive nitrite and sulfite detection in milk based on acid-treated Fe3O4@SiO2 nanoparticles.

In this work, a simple electrochemical sensing platform based on acid-treated Fe3O4@SiO2 nanoparticles was successfully prepared for nitrite and sulfite detection. Fe3O4@SiO2 nanoparticles were synthesized through the sol-gel and hydrothermal methods. Fe3O4@SiO2 presented positive charges after acid treatment, which could enhance the electrostatic attraction between Fe3O4@SiO2 and nitrite and sulfite. The Fe3O4@SiO2(acid-treated) modified magnetic glassy carbon electrode (MGCE) was applied to detect nitrite and sulfite using differential pulse voltammetry and cyclic voltammetry. Under optimized conditions, the developed electrochemical sensor presented good analytical properties for nitrite and sulfite detection with detection limits of 3.33 µmol/L and 31.57 µmol/L, respectively. The good recoveries varied from 85.18% to 111.02%, with a relative standard deviation of 0.23-4.80%. Furthermore, the Fe3O4@SiO2(acid-treated) modified MGCE showed better selectivity, reproducibility, and repeatability in nitrite and sulfite detection. Therefore, this proposed electrochemical sensor provides a new method for developing a nitrite and sulfite detection sensor.

Current analytical strategies for the determination of quinolone residues in milk.

Quinolones are potent antibacterial drugs extensively utilized for treating bacterial infections in poultry. However, the presence of quinolone antibiotic residues in milk is a matter of concern due to potential health risks and adverse effects on milk quality. This review provides an overview of current analytical strategies for the determination of quinolone residues in milk. Various sample preparation techniques, such as liquid-phase extraction, solid-phase extraction and QuEChERS, are discussed, along with detection methods including instrument-based detection, immune-based detection, and microbial detection. The advantages and limitations of each method are highlighted, as well as their applicability in different stages of milk production. Additionally, recent advancements in sample preparation and detection methods are presented. This comprehensive review aims to contribute to the development of accurate and reliable methods for the detection of quinolone residues in milk, ensuring the safety and quality of dairy products.

Label-Free quantitation of milk oligosaccharides from different mammal species and heat treatment influence.

Milk oligosaccharides (MOs) exhibit significant variations in concentrations and patterns among different species. However, there is limited knowledge about milk oligosaccharides in domestic animals and the impact of heat treatment on them. Here, we developed an LC-ESI-MS/MS method to analyze 11 milk oligosaccharides in 7 distinct species simultaneously. The results showed that human milk presented a completely different composition pattern of milk oligosaccharides from animals. In detail, animal milk predominantly contained sialylated oligosaccharides, and human milk had high levels of fucosylated neutral oligosaccharides. Notably, sheep milk exhibited similarities to human milk in terms of oligosaccharides composition. Then, the milk samples from dairy cows were treated with two common industrial heat treatments. We found that 65 °C treatment had no significant effect on the concentration of milk oligosaccharides, whereas 135 °C heating was associated with their decline, suggesting that high temperatures should be avoided in the processing of oligosaccharides supplemented/enriched products.

Movements of moisture and acid in gastric milk clots during gastric digestion: Spatiotemporal mapping using hyperspectral imaging.

Ruminant milk is known to coagulate into structured clots during gastric digestion. This study investigated the movements of moisture and acid in skim milk clots formed during dynamic gastric digestion and the effects of milk type (regular or calcium-rich) and the presence/absence of pepsin. We conducted hyperspectral imaging analysis and successfully modelled the moisture contents based on the spectral information using partial least squares regression. We generated prediction maps of the spatiotemporal distribution of moisture within the samples at different stages of gastric digestion. Simultaneously to acid uptake, the moisture in the milk clots tended to decrease over the digestion time; this was significantly promoted by pepsin. Moisture mapping by hyperspectral imaging demonstrated that the high and low moisture zones were centralized within the clot and at the surface respectively. A structural compaction process promoted by pepsinolysis and acidification probably contributed to the water expulsion from the clots during digestion.

Isolation and characterization of Bacillus cereus bacteriophage DZ1 and its application in foods.

Bacillus cereus is a pathogenic bacterium that causes food contamination, resulting in food poisoning such as diarrhea and emesis. Therefore, it is crucial to develop effective strategies to control this bacterium. In this study, we isolated and characterized a novel B. cereus phage, named DZ1. Morphological and genomic analyses revealed that phage DZ1 is a new species belonging to the Andromedavirus genus. Phage DZ1 was tolerant to a wide range of pH values (5-9), temperatures (4-55 â„ƒ), and high concentrations of NaCl solution (1000 mM). B. cereus with 21 different sequence types (STs) can be lysed by phage DZ1. Importantly, phage DZ1 inhibited B. cereus growth in spiked rice substrates or milk up to 36 and 72 h, respectively, with suppression of 3 log. Therefore, phage DZ1 is a useful biocontrol agent for the control of B. cereus in the food industry.

Intakes of Added Sugars, with a Focus on Beverages and the Associations with Nutrient Adequacy in US Adults (NHANES 2003-2018).

The Dietary Guidelines for Americans recommend adults increase their intake of nutrients that are under-consumed while limiting their intake of added sugars, sodium, and saturated fats. The purpose of this study was to examine the relationship between added sugars intake from specific types of beverages with added sugars (soft drinks, fruit drinks, sports and energy drinks, coffee and tea, and flavored milk) and nutrient adequacy among US adults (19+ y). Data from eight consecutive 2-y cycles of NHANES were combined (2003-2004 through 2017-2018), and regression analysis was conducted to test for trends in quantiles of added sugars intake from each beverage source and the rest of the diet (excluding those beverages) and nutrient adequacy. Results revealed significant associations that varied in direction according to the added sugars source, negative for some (i.e., soft drinks) in terms of greater percentages of adults not meeting a defined threshold of nutrient adequacy with higher added sugars intakes, and positive for others (i.e., fruit drinks, flavored milk, the rest of the diet) in terms of lower percentages of adults not meeting nutrient thresholds. In conclusion, the contribution of different added sugars sources to nutrient intakes is a critical consideration in developing population-based dietary recommendations.