Influence of beeswax-based fish oil oleogels on the mechanism of water and oil retention in Pacific white shrimp (Litopenaeus vannamei) meat emulsion gels: Filling, emulsification and phase transition.

Oleogels have been used in the gelled surimi products to replace animal fats due to its structure characteristics. The effect of structure characteristics in fish oil oleogels on the mechanism of oil/water retention was investigated in meat emulsions. Beeswax assembly improved the oil and water retention. The unsaturation degree of fatty acids lowered the mobility of bound water, immobilized water as well as bound fat in the fish oil oleogel, but enhanced the mobility of free water and protons of unsaturated fatty acids. Beeswax addition and oil phase characteristics could enhance ß-sheets, disulfide bonds and hydrophobic force to improve the viscoelasticity, gel strength and oil/water retention. Beeswax assembly facilitated the tight micro-sol network and filling effect, and high unsaturation degree promoted the emulsification effect, thus reducing phase transition temperature and juice loss. The study could lay the foundation for development of gelled shrimp meat products with EPA and DHA.

Based on proteomics probing into the deterioration mechanism of pork batter gel caused by different cooking temperatures.

The purpose of this experiment was to explore the influence of different cooking temperatures on the deterioration characteristics of pork batter gel by using proteomics, gel electrophoresis, size and chemical bond of aggregates. The results showed that the protein molecules of the pork batter gel was degraded during heating cooking and the protein aggregates were composed of many degraded protein fragments; compared with the control group 75 °C (0 min), the significant degradation of cytoskeleton showed at 110 °C (30 min) and 121 °C (30 min) and the significant degradation of myosin complexonly appeared at 121 °C (30 min). As the heating temperature points increased, compared with the control group 75 °C (0 min), the different temperatures could promote the separation of metal ions with proteins especially at 110 °C (30 min) and 121 °C (30 min), which could ultimately influence quality of pork batter gel by the size of particle. As the increase of heating temperature points, the recombination of aggregates composed of different proteins was not conducive to the retention of capillary water, which reduced the texture of pork batter gel. This research provided theoretical support for improving the process property of the meat products.

Alterations in gut microbiota and inflammatory cytokines after administration of antibiotics in mice.

Antibiotics are widely used to treat bacterial infection and reduce the mortality rate, while antibiotic overuse can cause gut microbiota dysbiosis. The impact of antibiotics on gut microbiota is not fully understood. In our study, four commonly used antibiotics (ceftazidime, cefoperazone-sulbactam, imipenem-cilastatin, and moxifloxacin) were given subcutaneously to mice, and their impacts on the gut microbiota composition and serum cytokine levels were evaluated through 16S rRNA analysis and a multiplex immunoassay. Antibiotic treatment markedly reduced gut microbiota diversity and changed gut microbiota composition. Antibiotic treatment significantly increased and decreased the abundance of Firmicutes and Bacteroidota, respectively. The antibiotic treatments increased the abundance of opportunistic pathogens such as Enterococcus and decreased that of Lachnospiraceae and Muribaculaceae. For moxifloxacin, the significantly high abundance of Enterococcus and Klebsiella was observed after 14 and 21 days of treatment. However, a relatively low abundance of opportunistic pathogens was found after 14 days of imipenem-cilastatin treatment. Additionally, the serum levels of various pro-inflammatory cytokines, such as IL-1ß, IL-12 (p70), and IL-17, significantly increased after 21 days of antibiotic treatments. Overall, these results provide a guide for rational use of antibiotics in clinical settings: short-term use of moxifloxacin is recommended with regard to gut microbiota health, and the 14-day use of imipenem-cilastatin may have a less severe impact than other antibiotics.IMPORTANCEAntibiotic treatments are directly associated with changes in gut microbiota and are effective against both pathogens and beneficial bacteria. Gut microbiota dysbiosis induced by antibiotic treatment could increase the risk of some diseases. Therefore, an adequate understanding of gut microbiota changes after antibiotic use is crucial. In this study, we investigated the effects of continuous treatment with antibiotics on gut microbiota, serum cytokines, and intestinal inflammatory response. Our results suggest that short-term use of moxifloxacin is recommended, and the 14-day use of imipenem-cilastatin may have a less severe effect on gut microbiota health than cefoperazone-sulbactam. These results provide useful guidance on the rational use of antibiotics with regard to gut microbiota health.

Optimizing ultrafast dynamic contrast-enhanced MRI scan duration in the differentiation of benign and malignant breast lesions.

OBJECTIVE: To determine the optimal scan duration for ultrafast DCE-MRI in effectively differentiating benign from malignant breast lesions. METHODS: The study prospectively recruited participants who underwent breast ultrafast DCE-MRI from September 2021 to March 2023. A 30-phase breast ultrafast DCE-MRI on a 3.0-T MRI system was conducted with a 4.5-s temporal resolution. Scan durations ranged from 40.5 s to 135.0 s, during which the analysis is performed at three-phase intervals, forming eight dynamic sets (scan duration [SD]40.5s: 40.5 s, SD54s: 54.0 s, SD67.5s: 67.5 s, SD81s: 81.0 s, SD94.5s: 94.5 s, SD108s: 108.0 s, SD121.5s: 121.5 s, and SD135s: 135.0 s). Two ultrafast DCE-MRI parameters, maximum slope (MS) and initial area under the curve in 60 s (iAUC), were calculated for each dynamic set and compared between benign and malignant lesions. Areas under the receiver operating characteristic curve (AUCs) were used to assess their diagnostic performance. RESULTS: A total of 140 women (mean age, 47 ± 11 years) with 151 lesions were included. MS and iAUC from eight dynamic sets exhibited significant differences between benign and malignant lesions (all p < 0.05), except iAUC at SD40.5s. The AUC of MS (AUC = 0.804) and iAUC (AUC = 0.659) at SD67.5s were significantly higher than their values at SD40.5s (AUC = 0.606 and 0.516; corrected p < 0.05). No significant differences in AUCs for MS and iAUC were observed from SD67.5s to SD135s (all corrected p > 0.05). CONCLUSIONS: Ultrafast DCE-MRI with a 67.5-s scan duration appears optimal for effectively differentiating malignant from benign breast lesions. CRITICAL RELEVANCE STATEMENT: By evaluating scan durations (40.5-135 s) and analyzing two ultrafast DCE-MRI parameters, we found a scan duration of 67.5 s optimal for discriminating between these lesions and offering a balance between acquisition time and diagnostic efficacy. KEY POINTS: Ultrafast DCE-MRI can effectively differentiate malignant from benign breast lesions. A minimum of 67.5-sec ultrafast DCE-MRI scan duration is required to differentiate benign and malignant lesions. Extending the scan duration beyond 67.5 s did not significantly improve diagnostic accuracy.

A Review of Plant-Based Drinks Addressing Nutrients, Flavor, and Processing Technologies.

Plant-based drinks have garnered significant attention as viable substitutes for traditional dairy milk, providing options for individuals who are lactose intolerant or allergic to dairy proteins, and those who adhere to vegan or vegetarian diets. In recent years, demand for plant-based drinks has expanded rapidly. Each variety has unique characteristics in terms of flavor, texture, and nutritional composition, offering consumers a diverse range of choices tailored to meet individual preferences and dietary needs. In this review, we aimed to provide a comprehensive overview of the various types of plant-based drinks and explore potential considerations including their nutritional compositions, health benefits, and processing technologies, as well as the challenges facing the plant-based drink processing industry. We delve into scientific evidence supporting the consumption of plant-based drinks, discuss their potential roles in meeting dietary requirements, and address current limitations and concerns regarding their use. We hope to illuminate the growing significance of plant-based drinks as sustainable and nutritious alternatives to dairy milk, and assist individuals in making informed choices regarding their dietary habits, expanding potential applications for plant-based drinks, and providing necessary theoretical and technical support for the development of a plant-based drink processing industry.

Ameliorating the stability of native/thermally denatured chicken-derived myofibrillar proteins particles in an aqueous system: The synergistic effect of acidification combined with inulin and inulin/sodium alginate.

The effect of acidification through hydrochloric acid combined with inulin (In), and inulin/sodium alginate (In/SA) on the stability of native/thermally denatured myofibrillar proteins (MPs/TMPs) particles in an aqueous system was investigated. At the same pH, MPs-In and TMPs-In particles were smaller and had higher absolute potentials than MPs-In/SA and TMPs-In/SA particles. Additionally, the size of MPs-In particles reached 1 µm, and the solubility increased from 21.73 ± 0.57 % to 76.26 ± 1.27 % when the pH was reduced from 5.0 to 3.0. The absolute potential of TMPs 3-In particles increased from 15.77 ± 0.72 to 28.20 ± 0.30 mV, and the solubility increased from 18.65 ± 0.72 % to 74.53 ± 0.74 %. Confocal laser microscopy revealed that, compared with pH 5.0 or 4.0, MPs-In/TMPs-In particles dispersed more evenly at pH 3.0 compared with pH 5.0 or 4.0. This further confirmed that electrostatic repulsion between particles maximally contributed to particle stability. Furthermore, the α-helix content in TMPs-In particles at pH 3.0 decreased from 41.51 ± 1.09 % (TMPs control) to 16.61 ± 1.87 %. This decrement of an up to 60 % led to decreased intramolecular hydrogen bonds and improved surface hydrophobicity. Therefore, a single polysaccharide (In) combined with MPs/TMPs particles exhibited higher dispersion and stability at pH 3.0. These findings could provide new insights into chicken-derived protein beverage processing.

Effects of protein aggregation induced by NaCl and temperature on gelation of silkworm (Antheraea pernyi) pupa raw powder.

Edible insects have great potential for producing protein-rich ingredients. This study aimed to investigate the effects of protein aggregation induced by NaCl (0-1 M) and temperature (65-95 °C) on gelation of Antheraea pernyi (A. pernyi) pupa raw powder. No thermal aggregates were observed at low temperature (65 °C), on the basis of there being no significant enhancement in turbidity and particle size (P > 0.05), regardless of NaCl concentrations. At elevated temperatures (75-95 °C), protein solutions exhibited significantly higher turbidity and particle size (P < 0.05), accompanied by an initial rise in surface hydrophobicity followed by a decline, alongside declining sulfhydryl. This marks the beginning of massive thermal aggregation driven by molecular forces. In addition, covalent (disulfide bonds) and non-covalent (hydrogen bonding, electrostatic interactions, and hydrophobicity) forces were influenced by NaCl, leading to variability in the protein aggregation and gelation. Correlation analysis indicates that the higher protein aggregation induced by ions was beneficial to the construction of more compact three-dimensional structures, as well as to the rheology, texture, and water-holding capacity of A. pernyi pupa gels. However, excessive salt ions destroyed the gel structure. Our findings will aid the use of A. pernyi pupae as textural ingredients in formula foods.

Regulation mechanism of curcumin-loaded oil on the emulsification and gelation properties of myofibrillar protein: Emphasizing the dose-response of curcumin.

The regulation mechanism of curcumin (CUR) in the oil phase on the emulsification and gelation properties of myofibrillar protein (MP) was investigated. CUR enhanced the emulsifying activity index (EAI) of MP but decreased its turbiscan stability index (TSI) and surface hydrophobicity, which exacerbated oil droplet aggregation. Medium amounts (200 mg/L) of CUR changed the 3D network architectures of emulsion gels from lamellar to reticular, improving the gels' water-holding capacity (WHC), storage modulus, springiness, and cohesiveness. Besides, the LF-NMR revealed that CUR had limited effects on the mobility of immobilized and free water. The α-helix of MP in gels with medium amounts of CUR decreased from 51% to 45%, but the ß-sheet increased from 23% to 27% compared to those without CUR. Overall, CUR has the potential to become a novel structural modifier in emulsified meat products due to its dose-response.

Polysaccharides, proteins, and their complex as microencapsulation carriers for delivery of probiotics: A review on carrier types and encapsulation techniques.

Probiotics provide several benefits for humans, including restoring the balance of gut bacteria, boosting the immune system, and aiding in the management of certain conditions such as irritable bowel syndrome and lactose intolerance. However, the viability of probiotics may undergo a significant reduction during food storage and gastrointestinal transit, potentially hindering the realization of their health benefits. Microencapsulation techniques have been recognized as an effective way to improve the stability of probiotics during processing and storage and allow for their localization and slow release in intestine. Although, numerous techniques have been employed for the encapsulation of probiotics, the encapsulation techniques itself and carrier types are the main factors affecting the encapsulate effect. This work summarizes the applications of commonly used polysaccharides (alginate, starch, and chitosan), proteins (whey protein isolate, soy protein isolate, and zein) and its complex as the probiotics encapsulation materials; evaluates the evolutions in microencapsulation technologies and coating materials for probiotics, discusses their benefits and limitations, and provides directions for future research to improve targeted release of beneficial additives as well as microencapsulation techniques. This study provides a comprehensive reference for current knowledge pertaining to microencapsulation in probiotics processing and suggestions for best practices gleaned from the literature.

A review of oil and water retention in emulsified meat products: The mechanisms of gelation and emulsification, the application of multi-layer hydrogels.

Emulsified meat products are key deep-processing products due to unique flavor and high nutritional value. Myosin dissolves, and protein aggregation and heat-induced gelation occur after myosin unfolds and hydrophobic groups are exposed. Myosin could form interfacial protein membranes and wrap fat globules. Emulsified fat globules may be filled in heat-induced gel networks. Therefore, this review intends to discuss the influences of heat-induced gelation and interfacial adsorption behavior on oil and water retention. Firstly, the mechanism of heat-induced gelation was clarified from the perspective of protein conformation and micro-structure. Secondly, the mechanism of emulsification stability and its factors affecting interfacial adsorption were demonstrated as well as limitations and challenges. Finally, the structure characteristics and application of multi-layer hydrogels in the gelation and emulsification were clarified. It could conclude that the characteristic morphology, spatial conformation and structure adjustment affected heat-induced gelation and interfacial adsorption behavior. Spatial conformation and microstructure were adjusted to improve the oil and water retention by pH, ionic strength, amino acid, oil phase characteristic and protein interaction. Multi-layer hydrogels facilitated oil and water retention. The comprehensive review of gelation and emulsification mechanisms could promote the development of meat products and improvement of meat processing technology.

Effects of tetrasodium pyrophosphate coupled with soy protein isolate on the emulsion gel properties of oxidative myofibrillar protein.

The effects of protein oxidation on the emulsion gel properties of myofibrillar protein (MP) in the presence of tetrasodium pyrophosphate (TSPP) and soybean protein isolate (SPI) were investigated from the perspective of interfacial protein interactions. The results showed that the emulsifying activity and emulsion stability of MP increased by 35.2 %-181.6 % with elevated H2O2 concentrations (1-20 mM), while the gel strength and water holding capacity of MP emulsions first increased to a maximum at 5 mM H2O2 and then decreased. TSPP and SPI further reinforced the effects caused by oxidation. The emulsifying properties of MP and its emulsion gel properties were closely related to surface hydrophobicity/hydrogen bonds/hydrophobic interactions and disulfide bonds among interfacial proteins, respectively. However, these correlations became difficult to define when TSPP and SPI were introduced. The study provides a theoretical basis for the strategy development to reduce protein oxidation damage on meat product quality.

Pickering emulsion stabilized by modified pea protein-chitosan composite particles as a new fat substitute improves the quality of pork sausages.

Pickering emulsion is a potential substitute for animal fat due to high stability and solid-like properties. Therefore, the effect of replacing 25%-100% pork backfat with Pickering emulsion (75% corn oil volume fraction) stabilized by modified pea protein-chitosan composite particles on the quality of sausages was studied. All meat pastes exhibited a strong gel-like rheological character (G' > G"). The incorporation of Pickering emulsion in sausages enhanced the textural properties (hardness, springiness, chewiness, cohesiveness and resilience) and the uniformity and compactness of micromorphology, as well as suppressed the cooking loss and TBARS content. In particular, the sausages with a backfat substitution ratio of 100%, showing a similar overall sensory acceptability to the backfat sausage, revealed the best rheological properties, texture properties and micromorphology and the lowest cooking loss and fat oxidation (P < 0.05). The results showed that Pickering emulsion stabilized by modified pea protein-chitosan composite particles is a potential fat substitute for meat products with the desirable characteristics.

Mechanism of 17ß-estradiol degradation by Rhodococcus equi via the 4,5-seco pathway and its key genes.

Steroid estrogens have been detected in oceans, rivers, lakes, groundwaters, soils, and even urban water supply systems, thereby inevitably imposing serious impacts on human health and ecological safety. Indeed, many estrogen-degrading bacterial strains and degradation pathways have been reported, with the 4,5-seco pathway being particularly important. However, few studies have evaluated the use of the 4,5-seco pathway by actinomycetes to degrade 17ß-estradiol (E2). In this study, 5 genes involved in E2 degradation were identified in the Rhodococcus equi DSSKP-R-001 (R-001) genome and then heterologously expressed to confirm their functions. The transformation of E2 with hsd17b14 reached 63.7% within 30 h, resulting in transformation into estrone (E1). Furthermore, we found that At1g12200-encoded flavin-binding monooxygenase (FMOAt1g12200) can transform E1 at a rate of 51.6% within 30 h and can transform E1 into 4-hydroxyestrone (4-OH E1). In addition, catA and hsaC genes were identified to further transform 4-OH E1 at a rate of 97-99%, and this reaction was accomplished by C-C cleavage at the C4 position of the A ring of 4-OH E1. This study represents the first report on the roles of these genes in estrogen degradation and provides new insights into the mechanisms of microbial estrogen metabolism and a better understanding of E2 degradation via the 4,5-seco pathway by actinomycetes.

The non-covalent interactions between whey protein and various food functional ingredients.

In daily diet, Whey protein (WP) is often coexisted with various Food functional ingredients (FFI) such as proteins, polyphenols, polysaccharides and vitamins, which inevitably affect or interact with each other. Generally speaking, they may be interact by two different mechanisms: non-covalent and covalent interactions, of which the former is more common. We reviewed the non-covalent interactions between WP and various FFI, explained the effect of each WP-FFI interaction, and provided possible applications of WP-FFI complex in the food industry. The biological activity, physical and chemical stability of FFI, and the structure and functionalities of WP were enhanced through the non-covalent interactions. The development of non-covalent interactions between WP and FFI provides opportunities for the design of new ingredients and biopolymer complex, which can be applied in different fields. Future research will further focus on the influence of external or environmental factors in the food system and processing methods on interactions.

Whey protein and xylitol complex alleviate type 2 diabetes in C57BL/6 mice by regulating the intestinal microbiota.

Type 2 diabetes (T2D) is a metabolic disorder that has become a major threat to public health. Epidemiological and experimental studies have suggested that whey protein isolate (WPI) and xylitol (XY) play an important role on T2D. This manuscript hypothesizes the supplementation of whey protein and xylitol complex (WXY) has the hypoglycemic and hyperlipidemia effect of T2D mice induced by the conjoint action of a high-fat diet and streptozotocin (STZ) by modulating of intestinal microbiota. The mice with diabetes displayed higher levels of fasting blood glucose (FBG), insulin, glycosylated hemoglobin, total triglycerides, total cholesterol, aspartate aminotransferase, alanine aminotransferase and other serum parameters than the normal mice. Treatment with WXY for 6 weeks significantly modulated the levels of FBG and insulin, improved insulin sensitivity, pancreas impairment and liver function in T2D mice, and the effect was better than that observed with WPI and XY groups. Moreover, supplementation with WXY significantly changed the diversity and composition of the intestinal microbiota in T2D mice and restored the intestinal bacteria associated with T2D (Firmicutes, Bacteroidetes, and Lactobacillus). This may be a potential mechanism for alleviating T2D symptoms. Spearman correlation analysis showed that the relative abundances of specific genera (Turicibacter, Lachnospiraceae_NK4A136_group, Lactobacillus, Candidatus_Saccharimonas, Faecalibaculum and Coriobacteriaceae_UCG-002) were correlated with the levels of blood glucose and serum parameters. Therefore, WXY may be considered a promising dietary supplement for T2D treatment in the future.

Tetrasodium pyrophosphate ameliorates oxidative damage to the TGase-catalyzed gelation of actomyosins.

The present study attempted to investigate the interactive roles of protein oxidation (0-20 mM H2O2) and tetrasodium pyrophosphate (TSPP) on the crosslinking efficiency of actomyosin mediated by transglutaminase (TGase). Oxidation at 0-20 mM H2O2 was not conducive to TGase-mediated crosslinking as indicated by the relative reduction of free amine consumption from 35.3% to 11.7%, and caused the principle crosslinking sites to progressively convert from myosin subfragment-1 (S1) to subfragment-1 (S2) as evidenced by electrophoresis. However, the binding of TSPP to myosin alleviated oxidation suppression to TGase-catalyzed crosslinking in varying degrees and retarded the migration of crosslinking site from S1 to S2. Moreover, oxidation (especially 20 mM H2O2) decreased the final (90 °C) elasticity index (EI) and water holding capacity of TGase-treated actomyosin gel, while TSPP intensified those of TGase-catalyzed actomyosin gel, indicating that TSPP had a positive effect on ameliorating the oxidative stress to TGase-catalyzed gelation of actomyosin.

Effect of fatty acid saturation on gel properties of salt-soluble protein in pork.

The objective of this study was to evaluate the effect of fatty acid saturation (oleic acid, linoleic acid, and linolenic acid) on water distribution, migration, and gel properties of pork salt-soluble protein, by detected indicators that are Low field nuclear magnetic resonance (LF-NMR), magnetic resonance imaging (MRI), water-holding capacity (WHC), and gel strength. These results suggested that the WHC and gel strength decreased with the decrease of fatty acid saturation (p < 0.05). LF-NMR analysis revealed that the relaxation time T21 and T22 decrease (p < 0.05) with the decrease of fatty acid saturation. Results also showed that the T21 increased and T23 decreased in linolenic acid group compared with oleic acid group. Meanwhile, the peak area ratio of P21 and P22 decreased (p < 0.05), while P23 increased (p < 0.05). Therefore, the saturation of fatty acids had a great influence on the gel properties of protein. PRACTICAL APPLICATION: It provides a theoretical basis for the production of polyunsaturated fatty acids emulsified gel meat products and promotes the development of meat processing industry.

Tetrasodium pyrophosphate promotes light meromyosin crosslinking by microbial transglutaminase.

Phosphates are commonly included in meat processing, where oxidation is inevitable, to improve water binding. This present study attempted to reveal the interactive roles of protein oxidation and tetrasodium pyrophosphate (TSPP) on the crosslinking pattern of myosin mediated by transglutaminase (TGase). Mild oxidation at 1 mM H2O2 facilitated the TGase-initiated crosslinking, with the dominate crosslinking site shifted from S1 (in nonoxidized myosin) to Rod. The introduction of TSPP alleviated the oxidation stress on proteins, and was conductive to the crosslinking reaction notably at the LMM domain. The crosslinking sites in untreated myosin were identified as Gln-613 (S1) and Gln-1498 (LMM) by amino-acid sequence analysis, while strongly oxidation resulted in the loss of Gln-1498. Contrastively, four new reactive crosslinking sites were generated by TSPP, one (Gln-558/Gln-567) located on S1 and three (Gln-1362, Gln-1374, and Gln-1423/Gln-1426) on LMM. Yet, Gln-1362 was eliminated under strong oxidation at 50 mM H2O2.

Characterization and comparison of lipids in bovine colostrum and mature milk based on UHPLC-QTOF-MS lipidomics.

Lipids in bovine milk have several biological activities, with implications for human health and the physical functionality of foods. However, alterations in the lipid profile of bovine milk during lactation are not well-studied. This study aimed to identify differences in lipids between bovine colostrum and mature milk, using a lipidomics approach. Using an advanced mass spectrometry-based quantitative lipidomics approach, 335 lipids assigned to 13 subclasses were characterized in bovine colostrum (BC) and mature milk (BM). In total, 63 significantly differential lipids (SDLs) were identified. Among the 63 SDLs, the levels of 21 lipids were significantly lower in BM than in BC, including 5 glycerophosphatidylethanolamines (PEs), 1 glycerophosphatidylglycerol (PG), and 15 triacylglycerols (TGs). The levels of the remaining 42 lipids increased in BM, including 1 cardiolipin (CL), 9 diacylglycerols (DGs), 9 dihexosylceramides (Hex2Cers), 3 hexosylceramides (HexCers), 3 glycerophosphatidic acids (PAs), 2 glycerophosphatidylcholines (PCs), 12 PEs, and 3 TGs. Furthermore, the correlations and related metabolic pathways of these 63 SDLs were analyzed to explore the mechanisms that alter bovine milk lipids during lactation. The seven most relevant pathways identified herein, ranked in accordance with their degree of influence on lactation, were glycerophospholipid metabolism, sphingolipid metabolism, glycerolipid metabolism, glycosylphosphatidylinositol-anchor biosynthesis, linoleic acid metabolism, alpha-linolenic acid metabolism, and arachidonic acid metabolism. Our results provide essential insights into mechanisms underlying alterations in bovine milk lipids during different lactation periods, along with practical information of specific nutrition and quality assessments for the dairy industry.

Determination of allosteric and active sites responsible for catalytic activity of delta 12 fatty acid desaturase from Geotrichum candidum and Mortierella alpina by domain swapping.

Cheese lacks essential fatty acids (EFAs). Delta 12 fatty acid desaturase (FADS12) is a critical enzyme required for EFA biosynthesis in fermentation of the predominant strains of cheese. Previously, we identified the FADS12 gene and characterized its function for the first time in Geotrichum candidum, a dominant strain used to manufacture soft cheese with white rind. In this study, we analyzed the molecular mechanism of FADS12 function by swapping domains from Mortierella alpina and G. candidum that had, respectively, high and low oleic acid conversion rates. The results revealed three regions that are essential to this process, including regions from the end of the second transmembrane domain to the beginning of the third transmembrane domain, from the end of the third transmembrane domain to the beginning of the fourth transmembrane domain, and from the 30-amino acid from the end of the sixth transmembrane domain to the C-terminal end region. Based on our domain swapping analyses, nine pairs of amino acids including H112, S118, H156, Q161, K301, R306, E307, A309 and S323 in MaFADS12 (K123, A129, N167, M172, T302, D307, I308, E310 and D324 in GcFADS12) were identified as having a significantly effect on FADS12 catalytic efficiency, and linoleic acid and its analogues (12,13-cyclopropenoid fatty acid) were found to inhibit the catalytic activity of FADS12 and related recombinant enzymes. Furthermore, the molecular mechanism of FADS12 inhibition was analyzed. The results revealed two allosteric domains, including one domain from the N-terminal region to the beginning of the first transmembrane domain and another from the 31st amino acid from the end of the sixth transmembrane domain to the C terminus. Y4 and F398 amino acid residues from MaFADS12 and eight pairs of amino acids including G56, L60, L344, G10, Q13, S24, K326 and L344 in MaFADS12 (while Y66, F70, F345, F20, Y23, Y34, F327 and F345 in GcFADS12) played a pivotal role in FADS12 inhibition. Finally, we found that both allosteric and active sites were responsible for the catalytic activity of FADS12 at various temperatures, pH, and times. This study offers a solid theoretical basis to develop preconditioning methods to increase the rate at which GcFADS12 converts oleic and linoleic acids to produce higher levels of EFAs in cheese.