Effects of pork sausage on intestinal microecology and metabolism in mice.

BACKGROUND: Processed meat, as an important part of the human diet, has been recognized as a carcinogen by the International Agency for Research on Cancer (IARC). Although numerous epidemiological reports supported the IARC's view, the relevant evidence of a direct association between processed meat and carcinogenicity has been insufficient and the mechanism has been unclear. This study aims to investigate the effects of pork sausage (as a representative example of processed meat) intake on gut microbial communities and metabolites of mice. Microbial communities and metabolites from all groups were analyzed using 16S rRNA gene sequencing and Ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometer (UPLC-Q-TOF/MS), respectively. RESULTS: The levels of Bacteroidetes, Bacteroides, Alloprevotella, Lactobacillus, Prevotella_9, Lachnospiraceae_NK4A136_group, Alistipes, Blautia, Proteobacteria, Firmicutes, Allobaculum, Helicobacter, Desulfovibrio, Clostridium_sensu_stricto_1, Ruminococcaceae_UCG-014, Lachnospiraceae_UCG-006 and Streptococcus (P < 0.05) were obviously altered in the mice fed a pork sausage diet. Twenty-seven metabolites from intestinal content samples and fourteen matabolites from whole blood samples were identified as potential biomarkers from multivariate analysis, including Phosphatidic acid (PA), Sphingomyelin (SM), Lysophosphatidylcholine (LysoPC), Diglyceride (DG), D-maltose, N-acylamides and so forth. The significant changes in these biomarkers demonstrate metabonomic variations in pork sausage treated rats, especially carbohydrate metabolism, lipid metabolism, and amino acid metabolism. CONCLUSION: The present study provided evidence that a processed meat diet can increase the risk of colorectal cancer and other diseases significantly by altering the microbial community structure and disrupting the body's metabolic pathways. © 2023 Society of Chemical Industry.

Systematic evaluation of the toxicological effects of deltamethrin exposure in zebrafish larvae.

Deltamethrin (DM) is a widely used pesticide and has been generally detected in aquatic systems. To systematically investigate the toxic effects, zebrafish embryos were treated with various concentrations of DM for 120 h. The LC50 was determined to be 102 µg L-1. Lethal concentrations of DM induced severe morphological defects in the surviving individuals. Under non-lethal concentrations, DM suppressed the development of neurons in the larvae, which was associated with the reduction in locomotor activity. DM exposure induced cardiovascular toxicity, including suppressed growth of blood vessels and enhanced heart rates. DM also disrupted the development of bones in the larvae. Moreover, liver degeneration, apoptosis and oxidative stress were observed in the larvae treated with DM. Correspondingly, the transcriptional levels of the genes related to the toxic effects were altered by DM. In conclusion, the results obtained in this study provided evidence that DM showed multiple toxic effects on aquatic organisms.

Extraction, Isolation and Identification of Low Molecular Weight Peptides in Human Milk.

Human milk contains numerous free low molecular weight peptides (LMWPs), which may play an important role in infant health and growth. The bioactivities of LMWPs are determined by their structures, especially the amino acid sequences. In the present study, 81 human milk samples were collected and purified by cation-exchange solid-phase extraction (SPE). Ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used for the separation and detection of free LMWPs in human milk. A total of 56 LMWPs were identified and quantified. These LMWPs were mainly derived from 3 regions of ß-casein, which were the amino acid fragments of 16-40, 85-110, and 205-226. The predominant LMWPs were RETIESLSSSEESITEYK, RETIESLSSSEESITEYKQKVEKVK, ETIESLSSSEESITEYK, TQPLAPVHNPIS, and QPLAPVHNPISV with molecular weights of 2247.9573, 2860.2437, 2091.8591, 1372.7666, and 1271.7212, respectively. The results indicated that the technique based on SPE and UPLC-QTOF-MS might greatly facilitate the analysis of LMWPs in human milk.

Efficient electrotransformation of Rhodococcus ruber YYL with abundant extracellular polymeric substances via a cell wall-weakening strategy.

Rhodococcus spp. have broad potential applications related to the degradation of organic contaminants and the transformation or synthesis of useful compounds. However, some Gram-positive bacteria are difficult to manipulate genetically due to low transformation efficiency. In this study, we investigated the effects of chemicals including glycine, isonicotinic acid hydrazide (INH), Tween 80 and penicillin G, as well as cell growth status, competent cell concentration, electroporation field strength, electroporation time and heat shock time, on the electrotransformation efficiency of the tetrahydrofuran-degrading bacterium Rhodococcus ruber YYL with low transformation efficiency. The highest electrotransformation efficiency was 1.60 × 106 CFU/µg DNA after parameter optimization. GmhD (D-glycero-D-manno-heptose 1-phosphate guanosyltransferase) gene, which is important in the biosynthesis of lipopolysaccharide, was deleted via the optimized electrotransformation method. Compared with wild-type strain, YYL ΔgmhD showed extremely high electrotransformation efficiency because the surface of it had no mushroom-like extracellular polymeric substances (EPS). In addition, the results showed that cell wall-weakening reagents might cause some translucent substances like EPS, to detach from the cells, increasing the electrotransformation efficiency of strain YYL. We propose that these results could provide a new strategy for unique bacteria that are rich in EPS, for which genetic manipulation systems are difficult to establish.

Whole-genome analysis of probiotic product isolates reveals the presence of genes related to antimicrobial resistance, virulence factors, and toxic metabolites, posing potential health risks.

BACKGROUND: Safety issues of probiotic products have been reported frequently in recent years. Ten bacterial strains isolated from seven commercial probiotic products on market were evaluated for their safety, by whole-genome analysis. RESULTS: We found that the bacterial species of three probiotic products were incorrectly labeled. Furthermore, six probiotic product isolates (PPS) contained genes for the production of toxic metabolites, while another three strains contained virulence genes, which might pose a potential health risk. In addition, three of them have drug-resistance genes, among which two strains potentially displayed multidrug resistance. One isolate has in silico predicted transferable genes responsible for toxic metabolite production, and they could potentially transfer to human gut microflora or environmental bacteria. Isolates of Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis are associated with low risk for human consumption. Based on a comparative genome analysis, we found that the isolated Enterococcus faecium TK-P5D clustered with a well-defined probiotic strain, while E. faecalis TK-P4B clustered with a pathogenic strain. CONCLUSIONS: Our work clearly illustrates that whole-genome analysis is a useful method to evaluate the quality and safety of probiotic products. Regulatory quality control and stringent regulations on probiotic products are needed to ensure safe consumption and protect human health.

Beneficial Effects of n-3 Polyunsaturated Fatty Acids on Offspring's Pancreas of Gestational Diabetes Rats.

We studied the long-term influence of gestational diabetes mellitus (GDM) on the pancreas of offspring and the effect of omega-3 polyunsaturated fatty acids (n-3 PUFAs) on offspring's pancreas. GDM offspring were divided into three groups: GDM offspring, n-3 PUFA-adequate-GDM offspring, and n-3 PUFA-deficient GDM offspring. All healthy and GDM offspring were fed up to 11 months old. The pancreas of GDM offspring exhibited fatty infiltration at 11 months old, whereas n-3 PUFA improved the pancreatic fatty infiltration. n-3 PUFA lowered the pancreatic oxidative stress and inflammation. Surprisingly, n-3 PUFA postponed pancreatic telomere shortening of GDM offspring at old age. Nontargeted metabolomics showed that many metabolites were altered in the pancreas of GDM offspring at old age, including l-valine, ceramide, acylcarnitines, tocotrienol, cholesteryl acetate, and biotin. n-3 PUFA modulated some altered metabolites and metabolic pathways. Therefore, GDM caused the long-term effects on offspring's pancreas, whereas n-3 PUFA played a beneficial role.

N-3 Polyunsaturated Fatty Acids Decrease Long-Term Diabetic Risk of Offspring of Gestational Diabetes Rats by Postponing Shortening of Hepatic Telomeres and Modulating Liver Metabolism.

The long-term influence of gestational diabetes mellitus (GDM) on offspring and the effect of omega-3 polyunsaturated fatty acids (n-3 PUFA) on GDM offspring are poorly understood. We studied the long-term diabetic risk in GDM offspring and evaluated the effect of n-3 PUFA intervention. Healthy offspring rats were fed standard diet (soybean oil) after weaning. GDM offspring were divided into three groups: GDM offspring (soybean oil), n-3 PUFA adequate offspring (fish oil), and n-3 PUFA deficient offspring (safflower oil), fed up to 11 months old. The diabetic risk of GDM offspring gradually increased from no change at weaning to obvious impaired glucose and insulin tolerance at 11 months old. N-3 PUFA decreased oxidative stress and inflammation in the liver of older GDM offspring. There was a differential effect of n-3 PUFA and n-6 PUFA on hepatic telomere length in GDM offspring. Non-targeted metabolomics showed that n-3 PUFA played a modulating role in the liver, in which numerous metabolites and metabolic pathways were altered when GDM offspring grew to old age. Many metabolites were related to diabetes risk, such as α-linolenic acid, palmitic acid, ceramide, oxaloacetic acid, tocotrienol, tetrahydro-11-deoxycortisol, andniacinamide. In summary, GDM offspring exhibited obvious diabetes risk at old age, whereas n-3 PUFA decreased this risk.

Changes in the soil microbial communities of alpine steppe at Qinghai-Tibetan Plateau under different degradation levels.

The alpine steppe at Qinghai-Tibetan Plateau is an important area for conserving water source and grassland productivity; however, knowledge about the microbial community structure and function and the risk to human health due to alpine plant-soil ecosystems is limited. Thus, we used prediction methods, such as Tax4Fun, and performed a metagenome pre-study using 16S rRNA sequencing reads for a small scale survey of the microbial communities at degraded alpine steppes (i.e., non-degraded (ND), lightly degraded (LD), moderately degraded (MD), heavily degraded (HD), and extremely degraded (ED) steppes) by Illumina high-throughput sequencing technology. Although there were no significant differences in the microbial alpha diversity among the different degraded alpine steppes and the dominant phyla at the different degraded alpine steppes, including Actinobacteria, Proteobacterial, Acidobacteria and Chloroflexi, were similar, the beta-diversity significantly differed, indicating that alpine steppe degradation might result in variation in microbial community compositions. The linear discriminate analysis (LDA) effect size (LEfSe) analysis found twenty-one biomarkers, most of which belonged to Actinobacteria, suggesting that microbes with a special function (such as the decomposition soil organic matter) might survive in alpine steppes. In addition, the functional profiles of the bacterial populations revealed an association with many human diseases, including infectious diseases. In addition, the microbial communities were mainly correlated with the populations of Gramineae and soil total phosphorous. These results suggested that alpine steppe degradation could result in variations in the microbial community composition, structure and function at Qinghai-Tibetan Plateau. Further studies investigating the degraded alpine steppe environment are needed to isolate these potential pathogenic microbes and help protect livestock using these alpine steppes.

Changes in the metabolite profile of breast milk over lactation stages and their relationship with dietary intake in Chinese women: HPLC-QTOFMS based metabolomic analysis.

The aim of the present study was to comprehensively evaluate the changes in the metabolite profile of breast milk over lactation stages and their relationship with dietary intake in Chinese women by HPLC-QTOFMS based metabolomic analysis. The colostrum, transitional milk and mature milk of thirty healthy lactating women were collected for analysis. Eighty-four differential metabolites over lactation stages were identified, including 12 fatty acyls, 15 glycerolipids, 23 glycerophospholipids, 7 sphingolipids, 7 vitamins, 5 nucleotides-related metabolites, 2 amino acids, 1 amino acid derivate, 9 dipeptides, 1 steroid hormone, 1 energy-related metabolite and 1 amine. Partial least-squares regression analysis indicated that the metabolite profiles of the colostrum, transitional milk and mature milk have a strong relationship with dietary intake (R2 values were 0.92, 0.87 and 0.74, respectively). However, among the 84 differential metabolites over lactation stages, only two showed a strong relationship with dietary intake: 1,24,25-(OH)3 vitamin D3 was positively correlated with the dietary intake of meat and eggs, protein and fat; 11ß-hydroxyprogesterone was negatively correlated with the dietary intake of fruit and carbohydrate. After adjusting for dietary intake, the variation trend of all the 84 differential metabolites over lactation stages remained unchanged. In conclusion, 84 differential metabolites in the breast milk of Chinese women over lactation stages were identified, and their variation trend was independent of dietary intake. These metabolites were partially different from those identified in previous metabolomic studies in the Western population. The present study is quite meaningful for understanding the variation of nutritional requirements in Chinese infants at different developmental stages and manufacturing optimal infant formulas for them.

Association of fatty acids and lipids metabolism in placenta with early spontaneous pregnancy loss in Chinese women.

The aim of the present study was to evaluate the association of fatty acids and lipids metabolism in placenta with early spontaneous pregnancy loss (ESPL) in Chinese women. Seventy women with ESPL and 29 healthy pregnant women who asked for legal induced abortion were included in the case and control groups, respectively. The gestational age of the subject foetuses in both the case and control groups ranged from 4 to 10 weeks. The total fatty acids composition in the decidual and villous tissues was detected by gas-liquid chromatography using a standard method. Metabonomics analysis of the decidual and villous tissues was conducted by ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOFMS). The total C18:3n-3 in the decidual and villous tissues, total n-3 polyunsaturated fatty acid (n-3 PUFA) in the decidual tissue, and total C18:2n-6 in the villous tissue were all significantly lower in the case group than in the control group. The ratio of C20:4n-6/C20:5n-3 in villous tissue was significantly higher, but prostaglandin I2 as well as hydroxyeicosapentaenoic acid, leukotriene B5 and thromboxane B3 in the villous tissue were significantly lower in the case group than in the control group. In addition, the low content of lysophosphatide in the decidual and villous tissues and the low content of diacylglycerol in the villous tissue were also associated with the occurance of ESPL. In conclusion, the lack of essential fatty acids, high ratio of C20:4n-6/C20:5n-3, abnormal eicosanoids metabolism and low content of lysophosphatide and diacylglycerol in the placenta were all potential risk factors for ESPL in Chinese.

Concentrations of estrogen and progesterone in breast milk and their relationship with the mother's diet.

The aim of the present study was to determine the concentrations of estradiol (E2), estriol (E3) and progesterone in breast milk over different lactation periods, and to assess their relationship with the mother's diet. Ninety-six breast milk samples as well as 24-hour dietary records from 32 lactating mothers were collected on day 1 (colostrum), day 14 (transitional milk) and day 42 (mature milk) after delivery in Hangzhou, China. The concentrations of E2, E3 and progesterone differed significantly through different periods of lactation (p < 0.001). The content of E2 in colostrum, transitional milk and mature milk was 1.60 ± 0.96 µg L-1, 0.83 ± 0.36 µg L-1 and 1.26 ± 0.48 µg L-1, respectively. The concentrations of E3 were 2.09 ± 1.66 µg L-1, 2.23 ± 1.74 µg L-1 and 4.64 ± 2.15 µg L-1, respectively. The concentrations of progesterone were 6.10 ± 8.30 µg L-1, 4.25 ± 4.76 µg L-1 and 1.70 ± 2.42 µg L-1, respectively. The concentration of progesterone in breast milk was significantly negatively correlated with the intake of protein (p = 0.015), fat (p = 0.008), vegetables (p = 0.012), and meat and eggs (p = 0.036), while the concentration of E3 was significantly positively correlated with the intake of soy products (p = 0.025). This information indicates that the concentrations of E2, E3 and progesterone in breast milk varied over the lactating period. Dietary intake will to some extent affect the contents of E3 and progesterone in breast milk.

Retinol and α-tocopherol in human milk and their relationship with dietary intake during lactation.

Human milk is considered as an optimal nutrition source for neonates, and vitamins A and E improve oxidative stress in offspring through breast milk. We aim to determine α-tocopherol and retinol contents in breast milk obtained over the course of lactation stages from mothers in three regions in China, and to investigate their correlation with maternal dietary intake during lactation. Human milk samples and 24 hour food records were obtained from mothers in Hangzhou (n = 34), Lanzhou (n = 34) and Beijing (n = 34) on day 1 (colostrum), day 14 (transitional milk) and day 42 (mature milk) postpartum. Concentrations of α-tocopherol and retinol in breast milk samples were measured by high-performance liquid chromatography. The breast milk concentrations of α-tocopherol and retinol decreased over progress of lactation stages (P < 0.001). The retinol content of transitional milk from Hangzhou was higher than that in Lanzhou (P = 0.019) and Beijing (P < 0.001), and the α-tocopherol content in colostrum from Hangzhou showed a lower concentration than that from Lanzhou (P = 0.001). There was a significantly positive correlation between retinol and α-tocopherol contents in colostrum (r = 0.37, P = 0.002), transitional (r = 0.41, P < 0.001) and mature milk (r = 0.53, P < 0.001). No significant correlation was found between any dietary constituents (grain, fruit, vegetable, dairy, egg and meat, oil and bean products) and either retinol or α-tocopherol.

Impact of 4-epi-oxytetracycline on the gut microbiota and blood metabolomics of Wistar rats.

The impact of 4-epi-oxytetracycline (4-EOTC), one of the main oxytetracycline (OTC) metabolites, on the gut microbiota and physiological metabolism of Wistar rats was analyzed to explore the dynamic alterations apparent after repeated oral exposure (0.5, 5.0 or 50.0 mg/kg bw) for 15 days as shown by 16S rRNA pyrosequencing and UPLC-Q-TOF/MS analysis. Both principal component analysis and cluster analysis showed consistently altered patterns with distinct differences in the treated groups versus the control groups. 4-EOTC treatment at 5.0 or 50.0 mg/kg increased the relative abundance of the Actinobacteria, specifically Bifidobacteriaceae, and improved the synthesis of lysophosphatidylcholine (LysoPC), as shown by the lipid biomarkers LysoPC(16:0), LysoPC(18:3), LysoPC(20:3), and LysoPC(20:4). The metabolomic analysis of urine samples also identified four other decreased metabolites: diacylglycerol, sphingomyelin, triacylglycerol, and phosphatidylglycerol. Notably, the significant changes observed in these biomarkers demonstrated the ongoing disorder induced by 4-EOTC. Blood and urine analysis revealed that residual 4-EOTC accumulated in the rats, even two weeks after oral 4-EOTC administration, ceased. Thus, through thorough analysis, it can be concluded that the alteration of the gut microbiota and disorders in blood metabolomics are correlated with 4-EOTC treatment.

Short-term toxicity assessments of an antibiotic metabolite in Wistar rats and its metabonomics analysis by ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

4-Epi-oxytetracycline (4-EOTC), one of main oxytetracycline (OTC) metabolites, can be commonly detected in food and environment. The toxicity and effects of OTC on animals have been well characterized; however, its metabolites have never been studied systemically. This study aims to investigate 15-day oral dose toxicity and urine metabonomics changes of 4-EOTC after repeated administration in Wistar rats at daily doses of 0.5, 5.0 and 50.0mg/kg bw (bodyweight). Hematology and clinical chemistry parameters, including white blood cell count, red blood cell count, total protein, globulin and albumin/globulin, were obviously altered in rats of 5.0 and 50.0mg/kg bw. Histopathology changes of kidney and liver tissues were also observed in high-dose groups. Urinary metabolites from all groups were analyzed using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Seventeen metabolites contributing to the clusters were identified as potential biomarkers from multivariate analysis, including aminoadipic acid, 6-phosphogluconate, sebacic acid, pipecolic acid, etc. The significant changes of these biomarkers demonstrated metabonomic variations in treated rats, especially lysine and purine metabolism. For the first time in this paper, we combined the results of toxicity and metabonomics induced by 4-EOTC for the serious reconsideration of the safety and potential risks of antibiotics and its degradation metabolites.

Biodegradation of di-n-butyl phthalate by a stable bacterial consortium, HD-1, enriched from activated sludge.

HD-1, a stable microbial consortium capable of mineralizing di-n-butyl phthalate (DBP), was developed from activated sludge. The dominant microorganisms in the consortium, Gordonia sp., Burkholderia sp. and Achromobacter sp., were identified by denaturing gradient gel electrophoresis (DGGE). The consortium could mineralize approximately 90% of 1200 mg/L DBP after 48 h of cultivation. The optimal DBP degradation conditions were 25-30 °C and pH 8.0-9.0. The addition of yeast (0.5 g/L), sodium acetate (0.5 g/L, 1.0 g/L), Brij 35 (0.2%, 0.5%, 1.0%), or Triton X-100 (0.2%) enhanced DBP degradation. The DBP degradation rate was influenced by the presence of dimethyl phthalate (DMP) and diethyl phthalate (DEP). Only one main intermediate, phthalic acid, could be monitored by gas chromatography-mass spectrometry (GC-MS) during the degradation process. The HD-1 consortium also utilized phenol, o-dihydroxybenzene as the sole carbon and energy source. The results indicate the consortium may represent a promising application for DBP bioremediation.

[Simultaneous determination of five yellow dyes in foods by high performance liquid chromatography coupled with tandem mass spectrometry].

A method based on high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) was developed for simultaneous determination of five yellow industrial dyes. The separations were performed with an Agilent ODS C18 column at a flow rate of 0.3 mL/min. The mobile phase was 5 mmol/L ammonium acetate (containing 0.1% formic acid)-acetonitrile (3:2, v/v). Under the optimized detection conditions, the linear ranges for Chrysoidine G and Basic Yellow 2 were 5.0-80.0 mg/L, and for Acid Orange I, Acid orange Il and Acid Yellow 36 were 10.0-160.0 microg/L. The limits of quantification for Chrysoidine G, Basic Yellow 2, Acid Orange I, Acid Orange II and Acid Yellow 36 were 20, 20, 40, 40 and 40 ng/g, respectively. The relative standard deviations of reproducibility of this method for retention time and peak area were no more than 0.50% and 2.14%, respectively. The method was applied to determine the recoveries of the above five dyes in chicken, bean products and yellow croaker were between 79.8%-95.2% with satisfactory results.

Oral administration activity determination of recombinant osteoprotegerin from silkworm larvae.

Osteoprotegerin (OPG) regulates the formation of osteoclasts and is involved in the regulation of bone resorption and remodeling. To investigate the feasibility of using silkworm (Bombyx mori) larvae to produce recombinant osteoprotegerin as a oral administration drug, the rh-OPG was expressed in the larvae of silkworm through the silkworm baculovirus expression system, and was orally administered to mice. Compared with the control, oral administration of rh-OPG was effective to decrease serum calcium concentration in normal mice, and block the bone loss induced by the loss of estrogen in ovariectomized mice. These results indicated that oral administration of rh-OPG expressed in silkworm larvae had the proper bioactivity.

[Expression and activity determination of TNFR domain of osteoprotegerin in E.coli and corresponding antibody preparation].

Osteoprotegerin (OPG) plays an important role in the regulation of bone resorption and remodeling. The TNFR domain of OPG, which is involved in the inhibition of formation and activity of osteoclasts, was amplified by PCR and inserted into multiple cloning site of PET-28a. The recombinant plasmid was transferred into E.coli BL21 to express recombinant protein. It was found that expressed product existed in the form of inclusion body. The inclusion body was solubilized, renatured and purified by affinity chromatography. Polyclonal antibodies with high specificity were obtained from the serum of rabbit immunized with purified recombinant protein. Mice were used to determine the hypocalcemic effect of the recombinant protein. Results showed that the recombinant protein expressed in E.coli had the proper bioactivity.

Expression and identification of mutated osteoprotegerin in culture cells and larvae of silkworm.

The mutated osteoprotegerin (OPG-372) gene was inserted into the baculovirus transfer vector pBacPAK8, and the recombinant plasmid was co-transfected with linearized Bm-BacPAK6 virus DNA into BmN cells, then homologous recombination occurred inside the cells. The recombinant virus BmNPV-OPG-372 was screened and identified by Southern blotting. The recombinant human OPG-372 was expressed in cultured cells and the larvae of silkworm by inoculation of recombinant virus. The expression products were run on the SDS-PAGE and their immunoreactivities were determined by Western blotting. It was found that a 42 kD recombinant protein was expressed in BmN cells and a 46 kD one in larvae respectively. The bioactivities of the recombinant proteins were determined by hypocalcemic effect assay in the mice. The results showed that the recombinant proteins had a significant hypocalcemic effect on mice sera.