Pig Bile Research Articles

A comparative study on the antipyretic effect and underlying mechanisms of different bile-fermented Arisaemas

Ethnopharmacological relevanceCattle bile Arisaema (CBA) and Pig bile Arisaema (PBA) are both processed products fermented from Arisaema erubescens (Wall.) Schott and animal bile, which are recorded in China Pharmacopoeia. Traditionally, bile Arisaema was often used for clearing heat and eliminating phlegm, calming wind and calming panic. Modern pharmacological researches suggest that both two drugs exert an antipyretic effect, while there is lack of the systematical and comparative evidence on underlying mechanism. Aim of the studyTo comprehensively clarify the differences and underlying mechanisms of antipyretic effect of the two drugs. MethodsIn this study, an accurate and reliable detection method based on ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-TQ MS) for comparing the content difference of bile acids from the two drugs was successfully established and applied. Besides, a dry yeast-induced fever rat model was established, and rectal temperature and content of pyrogenic cytokines were conducted to evaluate the antipyretic effect of CBA and PBA. Serum and hypothalamus untargeted metabolomics analysis based on ultra-performance liquid chromatography coupled with quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS/MS) technology were performed for elucidating the changes of metabolic profile. ResultsThe results indicated that CBA and PBA both exerted a significantly antipyretic effect, but CBA showed the characteristic of quicker onset and longer duration than that of PBA. The ELISA and western blotting analysis exhibited that the underlying antipyretic mechanism of the two drugs was closely associated with inhibiting inflammation through regulating TLR4/NF-κB signaling pathway. Moreover, the metabolism pathway analysis revealed that lipid metabolism and amino acid metabolism were greatly disturbed, which showed a certain correlation with antipyretic effect of two drugs. ConclusionCollectively, our results delineate a potential mechanism of two different bile Arisaemas against febrile via regulating metabolic disorders and targeting inhibition of inflammation for the improvement of fever symptom of the body. Notably, our current study suggested that CBA might be a better choice for suppressing fever clinically.

Comprehensive Analysis of Bile Medicines Based on UHPLC-QTOF-MSE and Machine Learning.

Based on UHPLC-QTOF-MSE analysis and quantized processing, combined with machine learning algorithms, data modeling was carried out to realize digital identification of bear bile powder (BBP), chicken bile powder (CIBP), duck bile powder (DBP), cow bile powder (CBP), sheep bile powder (SBP), pig bile powder (PBP), snake bile powder (SNBP), rabbit bile powder (RBP), and goose bile powder (GBP). First, 173 batches of bile samples were analyzed by UHPLC-QTOF-MSE to obtain the retention time-exact mass (RTEM) data pair to identify bile acid-like chemical components. Then, the data were modeled by combining support vector machine (SVM), random forest (RF), artificial neural network (ANN), gradient boosting (GB), AdaBoost (AB), and Naive Bayes (NB), and the models were evaluated by the parameters of accuracy (Acc), precision (P), and area under the curve (AUC). Finally, the bile medicines were digitally identified based on the optimal model. The results showed that the RF model constructed based on the identified 12 bile acid-like chemical constituents and random forest algorithm is optimal with ACC, P, and AUC > 0.950. In addition, the accuracy of external identification verification of 42 batches of bile medicines detected at different times is 100.0%. So based on UHPLC-QTOF-MSE analysis and combined with the RF algorithm, it can efficiently and accurately realize the digital identification of bile medicines, which can provide reference and assistance for the quality control of bile medicines. In addition, hyodeoxycholic acid, glycohyodeoxycholic acid, and taurochenodeoxycholic acid, and so forth are the most important bile acid constituents for the identification of nine bile medicines.

Rapid separation of bile acid isomers via ion mobility mass spectrometry by complexing with spiramycin.

Bile acid (BA) is one of the main active components of bile and has multiple isomers, the structure or content of its isomers often changes due to diseases and other health problems; thus, the accurate detection of BA isomers is very important. In this study, two groups of BA isomers of glycine-conjugated BAs and taurine-conjugated BAs were simultaneously separated and quantitatively analyzed by ion mobility mass spectrometry (IM-MS). Especially, baseline mobility separation between the isomers was achieved by the formation of binary complexes via simple interaction with spiramycin (SPM), for which a separation resolution (Rp-p) of 1.96was reached. Moreover, BA isomers were quantitatively analyzed, and the limit of detection (LOD) of absolute quantification for TCDCA/TUDCA and GUDCA/GCDCA/GHDCA was 0.514 and 0.611ng∙mL-1, respectively; the LODs for molar ratio ranges of relative quantification for TCDCA/TUDCA, GUDCA/GHDCA, and GCDCA/GHDCA were 1:18-30:1, 1:18-21:1, and 1:19-21:1, respectively. Additionally, BA isomers analyzed in pig bile powder and bear bile powder were measured, which were in good consistency with those labeled, revealing the differences in BA composition and content between the two powders. Finally, BA detection and recovery analyses were performed on serum samples, with a recovery rate of ≥73.69%, RSD of ≤6.8%, and SR (standard deviation of recoveries, the degree of difference between measured values and average recovery) of ≤1.27. Due to the simple, rapid, and lack of need for complex sample preparation and chromatographic separation, the proposed method can be an effective method for BA detection in practical samples.

Metabolic Engineering of Escherichia coli for Production of a Bioactive Metabolite of Bilirubin.

Bilirubin (BR) is an important ingredient of a valuable Chinese medicine, Calculus bovis. Over recent decades, increasing evidence has confirmed that BR offers health benefits in cardiovascular health, stroke, diabetes, and metabolic syndrome. However, BR is mainly produced by extraction from pig bile. In this study, we assembled an efficient pathway for BR production by metabolic engineering of Escherichia coli. First, heme oxygenase (HO1) and biliverdin reductase were co-expressed in E. coli. HPLC and LC-MS confirmed the accumulation of BR in the recombinant E. coli cells. To improve BR production, the catalytic abilities of HO1 from different species were investigated. In addition, the outermembrane-bound heme receptor (ChuA) and the enzymes involved in heme biosynthesis were overexpressed among which ChuA, 5-aminolevulinic acid dehydratase (HemB), protoporphyrin oxidase (HemG), and ferrochelatase (HemH) were found to enhance BR accumulation in E. coli. In addition, expression of ferredoxin (Fd) was shown to contribute to efficient conversion of heme to BR in E. coli. To increase supply of NADPH, isocitrate dehydrogenase (IDH), NAD kinase (nadK), NADP-specific glutamate dehydrogenase (gdhA), and glucose-6-phosphate 1-dehydrogenase (ZWF) were overexpressed and were found to enhance BR accumulation when these proteins were expressed with a low-copy plasmid pACYCduet-1. Modular optimization of the committed genes led to a titer of 17.2 mg/L in strain M1BHG. Finally, fed-batch fermentation was performed for the strains M1BHG and M1, resulting in accumulation of 75.5 mg/L and 25.8 mg/L of BR, respectively. This is the first report on biosynthesis of BR through metabolic engineering in a heterologous host.

Integrated metabolomics and network pharmacology analysis to explore pig bile-processed Rhizoma Coptidis and Fructus Evodiae sauce-processed Rhizoma Coptidis in lipopolysaccharide-induced inflammatory response

Pig bile- and Fructus Evodiae sauce-processed Rhizoma Coptidis (Danhuanglian, DHL; Yuhuanglian, YHL, respectively) are two types of processed Rhizoma Coptidis (Huanglian, HL) in traditional Chinese medicine (TCM). DHL and YHL are representative of HL generated from the subordinate and counter system processing methods, respectively, both noted for their anti-inflammatory effects. How these processing methods can affect the medicinal efficacy of HL remains a hot topic. Here, we discussed the influence of the two methods on the efficacy of final HL products (i.e., DHL and YHL) by comparing their components and anti-inflammatory mechanisms. Enzyme-linked immunosorbent assay was employed to measure inflammatory factors in RAW264.7 cells induced by lipopolysaccharide, and UPLC-Q-Exactive Orbitrap-MS was utilized to analyze the endogenous differential metabolites of RAW264.7 cells treated with HL, YHL, and DHL, and thus to identify the related metabolic pathways. Finally, using network pharmacology, we constructed a “disease-target-differential metabolites-active ingredients” network map. Compared with the control, all three products, HL, YHL, and DHL, significantly reduced IL-6, TNF-α, and IL-1β levels. 12 differential metabolites related to inflammation were identified and 25 target proteins were overlapping among the three groups. Notably, the anti-inflammatory effects of DHL and YHL were mediated by metabolic pathways such as aminoacyl-tRNA biosynthesis, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, and arginine biosynthesis. Specifically, DHL significantly impacted free fatty acid levels, which was not observed with HL and YHL. On screening, DHL had 9 active ingredients, including three from pig bile, and YHL had 12 active ingredients, with six from the processing excipient Fructus Evodiae. The distinct anti-inflammatory mechanisms and material basis of YHL and DHL were characterized by consistency and distinctiveness. Thus, this study underscores the significant influence of processing methods on the medicinal efficacy of TCMs by revealing their regulatory mechanisms and material bases.

Purification of Hyodeoxycholic Acid and Chenodeoxycholic Acid from Pig Bile Saponification Solution Using Column Chromatography

Purification of Hyodeoxycholic Acid and Chenodeoxycholic Acid from Pig Bile Saponification Solution Using Column Chromatography

Screening, Identification and Physiological Characteristics of Lactobacillus rhamnosus M3 (1) against Intestinal Inflammation.

The probiotic role of lactic acid bacteria (LAB) in regulating intestinal microbiota to promote human health has been widely reported. However, the types and quantities of probiotics used in practice are still limited. Therefore, isolating and screening LAB with potential probiotic functions from various habitats has become a hot topic. In this study, 104 strains of LAB were isolated from and identified in traditionally fermented vegetables, fresh milk, healthy infant feces, and other environments. The antibacterial properties-resistance to acid, bile salts, and digestive enzymes-and adhesion ability of the strains were determined, and the biological safety of LAB with better performance was studied. Three LAB with good comprehensive performance were obtained. These bacteria had broad-spectrum antibacterial properties and good acid resistance and adhesion ability. They exhibited some tolerance to pig bile salt, pepsin, and trypsin and showed no hemolysis. They were sensitive to the selected antibiotics, which met the required characteristics and safety evaluation criteria for probiotics. An in vitro fermentation experiment and milk fermentation performance test of Lactobacillus rhamnosus (L. rhamnosus) M3 (1) were carried out to study its effect on the intestinal flora and fermentation performance in patients with inflammatory bowel disease (IBD). Studies have shown that this strain can effectively inhibit the growth of harmful microorganisms and produce a classic, pleasant flavor. It has probiotic potential and is expected to be used as a microecological agent to regulate intestinal flora and promote intestinal health. It can also be used as an auxiliary starter to enhance the probiotic value of fermented milk.

An integrated strategy combining metabolomics and machine learning for the evaluation of bioactive markers that differentiate various bile.

Animal bile is an important component of natural medicine and is widely used in clinical treatment. However, it is easy to cause mixed applications during processing, resulting in uneven quality, which seriously affects and harms the interests and health of consumers. Bile acids are the major bioactive constituents of bile and contain a variety of isomeric constituents. Although the components are structurally similar, they exhibit different pharmacological activities. Identifying the characteristics of each animal bile is particularly important for processing and reuse. It is necessary to establish an accurate analysis method to distinguish different types of animal bile. We evaluated the biological activity of key feature markers from various animal bile samples. In this study, a strategy combining metabolomics and machine learning was used to compare the bile of three different animals, and four key markers were screened. Quantitative analysis of the key markers showed that the levels of Glycochenodeoxycholic acid (GCDCA) and Taurodeoxycholic acid (TDCA) were highest in pig bile; Glycocholic acid (GCA) and Cholic acid (CA) were the most abundant in bovine and sheep bile, respectively. In addition, four key feature markers significantly inhibited the production of NO in LPS-stimulated RAW264.7 macrophage cells. These findings will contribute to the targeted development of bile in various animals and provide a basis for its rational application.

Development history and prospect of Fel Ursi

Fel Ursi(bear bile) has been used as medicine for a long history, with wide clinical applications and definite curative efficacy. Fel Ursi has good pharmacodynamic activities in the treatment of liver and gallbladder diseases, and cardiovascular and cerebrovascular diseases. According to the places of origin, traditional Fel Ursi is divided into "Dongdan" and "Nandan". According to the gallbladder properties, it is divided into "Jindan" "Tiedan" "Caihuadan", and "Youdan". With the development of bear bile drainage technology, Pulvis Fellis Ursi has entered the market and been used clinically instead of Fel Ursi. At present, obtaining artificial Pulvis Fellis Ursi by chemical compounding and biotransformation is the hotspot in medical research, which can solve the shortage problem of bear bile resources, and also protect endangered animals. The quality problem of Fel Ursi in the market is prominent, with counterfeit products prepared from sheep bile, cow bile, pig bile, and chicken bile. Due to the scarcity of bear bile resources, the related research contents are scattered, and there is a lack of systematic analysis and summary. This paper focused on the development of Fel Ursi to clarify the source and classification of traditional Fel Ursi, and summarized the harvesting, processing, identification, and use of Fel Ursi. Additionally, the paper also compared the quality standards of Fel Ursi, summarized the technological development process of Pulvis Fellis Ursi, and prospected the modern research and clinical application of Pulvis Fellis Ursi, which is expected to provided references for the collation of bear bile resources, the clinical application of Fel Ursi, the development of Fel Ursi drugs, and related research on artificial Pulvis Fellis Ursi.

A composite scaffold fabricated with an acellular matrix and biodegradable polyurethane for the in vivo regeneration of pig bile duct defects

Bile duct regeneration is urgently needed to restore the normal function of the damaged biliary system. In this study, an artificial bile duct (ABD) was fabricated for extrahepatic bile duct regeneration based on biodegradable polyurethane (BPU) and ureter acellular matrix (UAM) to endow it with favorable biocompatibility and eliminate bile leakage during in vivo bile duct regeneration. The mechanical properties, in vitro simulation of bile flow and cytocompatibility of BPU-UAM ABD were evaluated in vitro, and surgical implantation in the biliary defect site in minipigs was implemented to reveal the in vivo degradation of BPU-UAM and regeneration of the new bile duct. The results indicated that BPU-UAM ABD with a mechanical strength of 11.9 MPa has excellent cytocompatibility to support 3T3 fibroblast survival and proliferation in extraction medium and on the scaffolds. The in vivo implantation of BPU-UAM ABD revealed the change of collagen content throughout the new bile duct regeneration. Biliary epithelial cells were observed at day 70, and continuous biliary epithelial layer formation was observed after 100 days of implantation. Altogether, the BPU-UAM ABD fabricated in this study possesses excellent properties for application study in the regeneration of bile duct. Statement of significanceExtrahepatic bile duct defects carry considerable morbidity and mortality because they are the only pathway for bile to go down into the intestinal tract. At present, no artificial bile duct can promote biliary regeneration. In this study, BPU-UAM ABD was built based on biodegradable polyurethane and ureter acellular matrix to form a continuous compact layer of polyurethane in the internal wall of UAM and avoid bile leakage and experimental failure during in vivo implantation. Our work verified the effectiveness of the synthesized biodegradable polyurethane emulsion-modified urethral acellular matrix in bile regeneration and continuous biliary epithelial layer formation. This study provided a new approach for the curing of bile duct defects and inducing new bile tissue formation.

Isolation, Identification, and Function of Rhodotorula mucilaginosa TZR2014 and Its Effects on the Growth and Health of Weaned Piglets.

A red yeast isolated from orange and grape soil and identified by the 26S rDNA sequence analysis revealed that it was Rhodotorula mucilaginosa and named TZR2014. Its biomass and carotenoid production reached a maximum when using the fermentation medium with pH 6.0, containing 5% glucose, 1% peptone, and 1.5% yeast powder. TZR2014 was resistant to 55°C for 15 min, 0.2% pig bile salts for 4 h, and artificial gastric and intestinal fluids. A total of thirty 28-day weaned pigs were divided into three groups, and the piglets were fed a basal diet (CON), a basal diet and orally administered 1 ml 1.0 × 1010 CFU/ml Candida utilis DSM 2361 three times (C. utilis), or a basal diet and orally administered 1 ml 1.0 × 1010 CFU/mL TZR2014 three times daily (R. mucilaginosa) for 4 weeks. Compared with the piglets in the CON group, those in the C. utilis or R. mucilaginosa group reported an increased average daily weight gain and average daily feed intake (P < 0.05) and a decreased feed/gain (P < 0.05). The diarrhea rate of piglets in the R. mucilaginosa group was lower than that in the CON and C. utilis groups (P < 0.05). Compared with that in the CON and C. utilis groups, the R. mucilaginosa group reported an increased ileum villus height (P < 0.05), serum concentration of total antioxidant content, total superoxide dismutase, and glutathione peroxidase and pepsin and lipase activities in the intestinal content, while it reported a decreased serum concentration of malondialdehyde and pH of the intestinal tract (P < 0.05). The relative abundances of Proteobacteria and Megasphaera of caecum in the R. mucilaginosa group were lower than those in the CON and C. utilis groups (P < 0.05). The relative abundances of Prevotella, Ruminococcaceae, Succinivibrio, Rikenellaceae RC9 gut group, and Roseburia of caecum in the R. mucilaginosa group were higher than those in the CON and C. utilis groups (P < 0.05). R. mucilaginosa TZR2014 can produce carotenoids and adapts to the animal's gastrointestinal environment. Oral R. mucilaginosa TZR2014 improved growth performance, enhanced antioxidant capacity, strengthened gastrointestinal digestion, and maintained the intestinal microbiological balance of piglets.

Biotransformation of Morphinone and Its Glutathione Adduct Derived from Morphine by Anaerobic Gut Microbes in Guinea Pigs.

Morphinone (MO) and its glutathione adduct (MO-GSH) are excreted into bile of guinea pigs after subcutaneous administration of morphine (M). In the present study, we examined metabolites of M in guinea pig feces. Surprisingly, minimal amounts of MO and MO-GSH were excreted into the feces, whereas dihydromorphine (DHM) and dihydromorphinone (DHMO), which are not found in bile of guinea pigs administered M, were detected in the feces. Incubation of MO and MO-GSH with the contents of the large intestine under anaerobic conditions resulted in their conversion into DHMO. These results suggest that MO-GSH undergoes C-S cleavage by gut microbes to form MO, which is anaerobically reduced to DHMO excreted into feces.

Rapid identification of bear bile powder from other bile sources using chip-based nano-electrospray ionization tandem mass spectrometry.

Bear bile powder (BBP) is a widely used traditional Chinese medicine (TCM), and bile acids (BAs) are the main active components in BBP. Due to the scarcity of BBP resources, adulterations often occur in the market. Conventional methods to distinguish them are usually complicated and time-consuming. To enhance effectiveness and accuracy, a rapid and rough analytical method is desperately needed. In this study, a rapid strategy using chip-based nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was established to distinguish BBP from other sources of bile powder (BP). In addition, the results were further verified by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry (UPLC/MS). The precision of the chip-based nano-ESI-MS/MS method was validated to be acceptable with relative standard deviation (RSD) <15%. The distinction between BBP and other sources of BP, including common adulterants of pig bile powder (PBP), cattle bile powder (CBP), sheep bile powder (SBP), and chicken bile powder (CkBP), can be observed in the spectra. By using orthogonal partial least-squares discriminant analysis (OPLS-DA), more potential m/z markers were investigated. A BAs-related m/z marker of 498.3 was discovered as a typical differential molecular ion peak and was identified as tauroursodeoxycholic acid (TUDCA) and taurochenodeoxycholic acid (TCDCA) in BBP. The proposed strategy has simple sample pretreatment steps and significantly shortened analysis time. As an emerging technology, chip-based nano-ESI-MS not only provides a reference for the rapid distinction of adulterated Chinese medicines, but also provides some insights into the identification of other chemicals and foods.

Cattle Bile Arisaema Aqueous Extracts Protect Against Febrile Seizures in Rats Through Regulating Neurotransmitters and Suppressing Neuroinflammation

Cattle bile Arisaema (CBA) is a traditional medicine used for the treatment of febrile seizures (FS) for thousands of years in China. However, its application is greatly limited due to cost reasons, and pig bile Arisaema (PBA) is the main commercial product instead. Additionally, the underlying mechanism of CBA for the treatment of FS still remains unknown. In this study, we investigated the anti-convulsant effect and potential mechanism of the CBA aqueous extract for the first time through a hot-water bath-induced FS rat model. Our results showed that pre-treatment with CBA dramatically lowered the incidence rate and generation times and prolonged the latency of FS. In addition, CBA effectively ameliorated neuronal damage and regulated neurotransmitter disorder induced by FS in the rat hippocampus. The enzyme-linked immunosorbent assay, western blotting, immunohistochemical, and qRT-PCR results exhibited that CBA suppressed the expression of GFAP, TLR4, NF-κB, HMGB1, NLRP3, TNF-α, IL-1β, and IL-6 and consequently inhibited the neuroinflammation induced by FS. Interestingly, although the CBA and PBA aqueous extracts possessed the same trend on the changes caused by FS, the improvement of FS by CBA is markedly better than that by PBA. These findings indicate that CBA exerts a protective effect on febrile seizures through regulating neurotransmitter disorder and suppressing neuroinflammation.

Production of bilirubin by biotransformation of biliverdin using recombinant Escherichia coli cells.

Bilirubin, a natural intermediate in heme degradation, is a valuable Chinese medicine used in more than 50 traditional Chinese medicine (TCM) preparations. At present, bilirubin is mainly produced by extraction from pig bile, but a shortage of the raw material has increased the price, to about US$10,000/kg in the Chinese market. Biliverdin, the precursor of bilirubin, is more abundant and less expensive than bilirubin, but it is not used in TCM. Thus, the biotransformation of biliverdin by biliverdin reductase (BvdR) may be a practical way to produce bilirubin. In this study, the codon-optimized gene of biliverdin reductase (mbvdR) from the cyanobacterium Synechocystis was cloned into Escherichia coli BL21(DE3), and the conditions for BL21-mBvdR expressing BvdR were optimized. Resting BL21-mBvdR cells were employed as biocatalysts to biotransform biliverdin to bilirubin. At a concentration of biliverdin substrate of 450mg/L in the reaction mixture, the bilirubin content in dry cells reached 20.8 ± 0.8mg/g, with a conversion yield of 72.3%. Therefore, recombinant E. coli expressing BvdR can be applied to biotransform biliverdin to bilirubin, providing a potential alternative process for bilirubin production.

Identification and Distribution of Sterols, Bile Acids, and Acylcarnitines by LC-MS/MS in Humans, Mice, and Pigs-A Qualitative Analysis.

Sterols, bile acids, and acylcarnitines are key players in human metabolism. Precise annotations of these metabolites with mass spectrometry analytics are challenging because of the presence of several isomers and stereoisomers, variability in ionization, and their relatively low concentrations in biological samples. Herein, we present a sensitive and simple qualitative LC–MS/MS (liquid chromatography with tandem mass spectrometry) method by utilizing a set of pure chemical standards to facilitate the identification and distribution of sterols, bile acids, and acylcarnitines in biological samples including human stool and plasma; mouse ileum, cecum, jejunum content, duodenum content, and liver; and pig bile, proximal colon, cecum, heart, stool, and liver. With this method, we detected 24 sterol, 32 bile acid, and 27 acylcarnitine standards in one analysis that were separated within 13 min by reversed-phase chromatography. Further, we observed different sterol, bile acid, and acylcarnitine profiles for the different biological samples across the different species. The simultaneous detection and annotation of sterols, bile acids, and acylcarnitines from reference standards and biological samples with high precision represents a valuable tool for screening these metabolites in routine scientific research.

Detection of hepatitis E virus RNA from pig bile collected at a slaughterhouse in Japan.

Hepatitis E virus (HEV) is a zoonotic pathogen that circulates mainly between pigs and humans. In Japan, the number of confirmed HEV cases has increased over the past decade, with the majority reported as domestic HEV infections. HEV-infected pork products may be associated with this increase, but there is limited information on HEV in pork in Japanese markets. From February to March 2020, gallbladders were collected from 200 slaughtered pigs shipped from 14 farms and were surveyed to detect HEV RNA in bile using reverse transcription quantitative polymerase chain reaction. The samples were then sequenced and genotyped. Twenty pigs were positive for HEV ribonucleic acid, and seven samples had Ct values of less than 30. Among these 20 pigs, virus strains from 14 pigs were determined as genotype 3. This report indicated that HEV-contaminated pork liver was shipped to consumer markets and demonstrated the importance of detection of HEV in meat ready for shipment.

A bacteriophage JN02 infecting multidrug‐resistant Shiga toxin‐producing Escherichiacoli: isolation, characterisation and application as a biocontrol agent in foods

AbstractBacteriophages are increasingly considered as novel biocontrol agents against Multidrug‐resistant (MDR) Shiga toxin‐producing Escherichia coli (STEC). In this study, a virulent phage JN02 capable of infecting MDR STEC was isolated, characterised and evaluating its efficacy in reducing MDR STEC in foods. One‐step growth and stability assay showed that JN02 had a relatively short latent period (15 min), large burst sizes (69 PFU/cell), and high physicochemical stability (temperature (30–60°C), pH (2–11), 0.1%, 1% pig bile salt, biocides and food additives (100% chloroform, 0.1% acetic acid, 1% potassium sorbate). In spot tests, 18 E. coli strains were sensitive to JN02. In efficiency of plating (EOP), 4 E. coli strains were sensitive to JN02. Electron microscopy and genome sequencing showed that JN02 belongs to the family Myoviridae. Importantly, no known virulence‐associated, lysogenic and antibiotic genes were identified in the genome of JN02. Viable cell counts of MDR STEC were significantly (P &lt; 0.05) reduced in milk (no viable counts) and beef samples (1.0–1.9 log10 CFU cm−2 reduction) when treated with JN02 at 4 °C. The results indicated that JN02 might be a new potential natural biological control agent for MDR STEC in foods.

Hyocholic acid and glycemic regulation: comments on ‘Hyocholic acid species improve glucose homeostasis through a distinct TGR5 and FXR signaling mechanism’

Hyocholic acid species (HCA, hyodeoxycholic acid, and their glycine and taurine conjugated forms) comprise 80% of the composition of pig bile (Haslewood, 1956). An interesting fact about pigs is that they do not get diabetes even though they eat almost anything and in abundant amounts, a diabetes-promoting diet. The first use of pig bile for treatment of 'xiao-ke', a condition known today as diabetes, was recorded ∼400 years ago by the Chinese medical practitioners in the Compendium of Materia Medica (Li, 1573‒1593). Recently, we found HCA species as novel biomarkers for metabolic diseases (Zheng et al., 2021b) and also identified the role of HCA species in the prevention of diabetes as well as their mechanism of action (Zheng et al., 2021a). Although bile acids (BAs) are mostly associated with their aid in food digestion, they have also been shown to act as signaling molecules by binding to two particular receptors, farnesoid X receptor (FXR) and the G-protein-coupled receptor, TGR5. Experiments were thus directed to the effect of HCA binding to these two BA receptors on glycemic regulation in both in vivo and in vitro models.

Isolation, Identification and Function of Pichia anomala AR2016 and Its Effects on the Growth and Health of Weaned Pigs.

Simple SummaryPichia anomala is a simple pseudomycelium with a round cell shape, product killer factors, β-1,3-glucanase, and phytase. P. anomala had the functions of anticorrosion, improving the nutritive value of feed, increasing the protein content, and reducing the concentration of nutritive substances. In this study, P. anomala AR2016 is isolated and identified from solid wine koji, and its culture conditions are optimized. Heat tolerance, bile salt tolerance, and artificial gastric and intestinal juice tolerance are evaluated. In our methodology, thirty weaned pigs were randomly divided into three groups with 10 barrows in each, and fed a maize-soybean meal diet and orally administered 0.85% saline (CK), 1 mL 1 × 109 cfu/mL Candida utilis (C. utilis), and 1 mL 1 × 109 cfu/mL P. anomala once daily for 28 days (P. anomala). Our results show that P. anomala AR2016 grew best in yeast extract peptone dextrose medium with pH 5.0 at 28 °C, 180 r/min and could tolerate 45 °C for 0.5 h, 0.2% pig bile salts, simulated gastric fluid, and 1.0% simulated intestinal fluid. Our study indicates that P. anomala AR2016 can tolerate high acidity and high bile salts, and has high survivability in the artificial gastric intestinal juice environment. In conclusion, oral administration of P. anomala AR2016 improves the growth performance, reduces the incidence of diarrhea, enhances intestinal barrier function, and improves microflora in weaned pigs.This study isolates and identifies Pichia anomala (P. anomala) AR2016, and studies its effect on the growth and health of weaned pigs. A P. anomala strain from solid wine koji is isolated and identified using 26S rDNA analysis, and its culture conditions are optimized. Heat tolerance, bile salt tolerance, artificial gastric, and intestinal juice tolerance are evaluated. In our methodology, thirty 28 d Large White × Landrace × Rongchang weaned pigs were randomly divided into three groups with 10 barrows in each, and fed a maize-soybean meal diet and orally administered 0.85% saline (CK), 1 mL 1 × 109 cfu/mL Candida utilis (C. utilis), and 1 mL 1 × 109 cfu/mL P. anomala once daily for 28 days. A P. anomala strain was identified and named P. anomala AR2016. P. anomala AR2016 grew best in yeast extract peptone dextrose medium with pH 5.0 at 28 °C, 180 r/min and could tolerate 45 °C for 0.5 h, 0.2% pig bile salts, simulated gastric fluid, and 1.0% simulated intestinal fluid. Our results show that compared with the CK group, orally administered P. anomala AR2016 increases average daily gain, the ileal villus height, the ileal mucosal concentrations of occludin and zonula occluens-1, the serum glucose and total protein concentration, total superoxide dismutase, glutathione peroxidase, and total antioxidative capacity activity, the trypsin and lipase activity in jejunal and ileal contents, the jejunal and ileal mucosa mRNA levels of ALP, TNF-α, and TLR-2, and the relative abundance of Bacteroidetes, Actinobacteria, Succinivibrionaceae, Lachnospiraceae, and Prevotellaceae (p < 0.05). Compared with the CK group, oral administration of P. anomala AR2016 decreased the incidence of diarrhea, aspartate aminotransferase activity, alanine amino-transferase-activity, malondialdehyde, D-lactic acid and endotoxin content in serum, the mRNA level of aminopeptidase N of ileum mucosa, and the relative abundance of Proteobacteria, Clostridiaceae, Campylobacteraceae, Vibrionaceae, Bacillus, and Pseudon (p < 0.05). Collectively, the study indicates that P. anomala AR2016 can tolerate high acidity and high bile salts, and has high survivability in the artificial gastric intestinal juice environment. Oral administration of P. anomala AR2016 improves the growth performance, reduces the incidence of diarrhea, enhances intestinal barrier function, and improves microflora in weaned pigs.