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.

Production and evaluation of enzyme-modified cheese adding protease or lipase to improve quality properties.

Enzyme-modified cheese (EMC) produced by enzyme hydrolysis is a natural, cost-effective, and flexible alternative to using natural cheese in industrial applications. The modification of cheese by enzymes can increase their benefits for consumer acceptance and health, and intensify the specific cheese flavor. We evaluated the properties of cheese with added protease (Ep) or lipase (El), including texture, sensory, organic acids, volatile compounds, and free amino acids. As results, the hardness and gumminess of the cheese reached their maximum values when the concentration of protease and lipase was 0.1% and 0.6%, respectively. Interestingly, the bitterness and astringency of the cheese was reduced. The highest scores for odor, taste, and overall acceptability were observed on 0.08% protease in Ep and 0.8% lipase in El. Compared with the anchor cheese, eight new compounds were produced after the addition of protease and nine new compounds were produced after the addition of lipase. Irrespective of the type of enzyme, the content of free amino acids decreased slightly with the increase in enzyme content. From the point of view of adding enzyme species, the free amino acids content of Ep was generally higher than that of El, and glutamic acid and proline contents were high. Acetic acid concentrations (aroma-active compounds) of enzyme-modified cheese using protease and lipase were 482-931 mg/100 g and 30-36 mg/100 g, respectively, which were significantly increased. According to the results obtained in this study, a cheese with higher sensorial and textural acceptability was obtained by adding the appropriate protease or lipase.

Characterization and shelf stability of sweetened condensed milk formulated with different sucrose substitutes during storage.

Sweetened condensed milk (SCM) is a value-added milk product with extended shelf life and high levels of nutrition. The high level of sucrose may lead to health problems. Many studies have focused on the reduction of sucrose but seldomly on different combination of sugar substitutes. This study aims to find an ideal sucrose substitution through physiochemical, microbiological and sensory properties of SCM under different storage times. The results demonstrated that substitution with 20% trehalose, 5% lactulose and 15% erythritol resulted in similar sensory and color as control group. The volatile flavor analysis showed that substitution with 30% trehalose, 5% lactulose and 5% polyols was the most similar and hexanoic acid was the symbolistic flavor. Sucrose replacement increased the antibacterial effect and Staphylococcus, Penicillium, Apiotrichum and Candida were widely present. Substitution with 30% trehalose, 5% lactulose and 5% polyols resulted in the most similar water activity, texture, aroma and microbial diversity.

Aptamer-conjugated graphene oxide-based surface assisted laser desorption ionization mass spectrometry for selective extraction and detection of Aß1-42 in an Alzheimer's disease SH-SY5 cell model.

The generation and accumulation of amyloid-beta peptide (Aß1-42) in amyloid plaques are key characteristics of Alzheimer's disease (AD); thus, specific detection of Aß1-42 is essential for the diagnosis and treatment of AD. Herein, an aptamer-conjugated graphene oxide (Apt-GO) sensor was synthesized by π-π and hydrophobic interactions using thiol poly (ethylene glycol) amine (SH-PEG-NH2) as a spacer unit. Then, it was applied to selective capture of Aß1-42, and the resulting complex was directly analyzed by surface-assisted laser desorption ionization mass spectrometry (SALDI-MS). The results revealed that the Apt-GO could enhance the detection specificity and reduce non-specific adsorption. This method was validated to be sensitive in detecting Aß1-42 at a low level in human serum (ca. 0.1 µM) within a linear range from 0.1 to 10 µM. The immobilizing amount of aptamer on the GO was calculated to be 36.1 nmol/mg (RSD = 11.5%). In conclusion, this Apt-GO-based SALDI-MS method was sensitive and efficient in selective extraction and detection of Aß1-42, which proved to be a good option for early AD diagnosis.

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.

Degradation kinetics and isomerization of 5-O-caffeoylquinic acid under ultrasound: Influence of epigallocatechin gallate and vitamin C.

5-O-Caffeoylquinic acid (5-CQA), also known as chlorogenic acid, exhibits various biological activities. Hence the interest in its property change during processing and extraction has increased. The present work studied the influence of ultrasound on the stability of 5-CQA at different pH (pH 4.69, 7.09, 7.69 and 9.22) in water and 50% methanol-water system. Different parameters including solvent, ultrasonic power, time, temperature, duty cycle, and liquid height were investigated during the treatment. Results indicate that ultrasound accelerated the degradation of 5-CQA. Based on Weibull model, the degradation kinetics were described. The rate constant (k) of the degradation increased with the increasing pH, demonstrating the alkali sensitivity of 5-CQA. The isomerization of 5-CQA to 3- and 4-O-caffeoylquinic acid was found at neutral and alkaline conditions, which was further boosted by ultrasound. The stability of 5-CQA was improved by adding epigallocatechin gallate (EGCG) and vitamin C (VC) respectively.

Determination of four nitrofuran metabolites in gelatin Chinese medicine using dispersive solid phase extraction and pass-through solid phase extraction coupled to ultra high performance liquid chromatography-tandem mass spectrometry.

This study established a validated analytical method for the first time on the determination of nitrofuran metabolites, including semicarbazide (SEM), 1-aminohydantoin (AHD), 3-amino-2-oxazolidinone (AOZ) and 3-amino-5-morpholinomethyl-2-oxazolinone (AMOZ) in gelatin Chinese medicine. A C18 column with the mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate in water was used to separate these nitrofuran metabolites. The limit of detection of SEM, AHD, AOZ and AMOZ were found to be 0.2 µg/kg, 0.3 µg/kg, 0.2 µg/kg and 0.2 µg/kg, whereas their limit of quantification were 0.6 µg/kg, 0.8 µg/kg, 0.6 µg/kg and 0.5 µg/kg. These nitrofuran metabolites exhibited a good linear standard curve (regression coefficients above 0.99) with a concentration range of 2 µg/L to 100 µg/L. Regarding extraction procedure, gelatin Chinese medicine was pre-treated with pepsin and then extracted using 5% formic acid (v/v) in acetonitrile. The resultant extract was purified through dispersive solid phase extraction using 1000 mg anhydrous sodium sulfate, 300 mg octadecyl carbon silica gel sorbent absorbent and 500 mg ethylenediamine-N-propyl carbon silica gel absorbent, and then further purified on Oasis PRiME HLB cartridges. The matrix effect was effectively eliminated after the clean-up procedure as confirmed by comparing the ratio of standard curves prepared by standards dissolved in both matrix solvent and 5 mmol/L ammonium acetate in water: acetonitrile (95:5, v/v). The recoveries of these nitrofuran metabolites under the 1 µg/kg, 2 µg/kg and 10 µg/kg spiking levels were between 77.4% and 95.6%. These metabolites after the extraction were stable at 4 °C for 24 h. The validated method was used to analyze the residue level of these nitrofuran metabolites in 25 gelatin Chinese medicines. Results showed that only one Colla Corii Asini sample contained SEM (2.52 µg/kg) and AOZ (6.27 µg/kg), whereas one Testudinis Carapacis et Plastri sample had SEM (1.27 µg/kg) and AMOZ (9.53 µg/kg).

Determination of Chloramphenicol, Thiamphenicol and Florfenicol in Chinese Gelatin Medicines using Dispersive Solid-Phase Extraction Coupled with Ultra High-Performance Liquid Chromatography-Mass Spectrometry.

This study established a rapid and reliable method to determine chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FF) residues in Chinese gelatin medicines. CAP, TAP and FF were extracted from medicine samples using 2% (v/v) ammonium hydroxide in acetonitrile. Trypsin was used to eliminate the matrix effect caused by protein components in gelatin medicines, whereas anhydrous sodium sulfate, C18-N and NH2-PSA adsorbents were applied to reduce matrix effect induced by other components. The analytical method of these drugs was optimized on ultra high-performance liquid chromatography-mass spectrometer (UHPLC-MS/MS) through the analysis of their standard linearity and regression. The optimized extraction and analytical method were validated in one Chinese gelatin medicine sample (Colla corii asini, E Jiao) with three fortification levels (2, 5 and 10 µg/kg), and the recoveries of these drug residues ranged of 87.6-102.7%. The limit of detection and quantification of CAP, TAP and FF in the sample were 0.2 and 0.5 µg/kg, 0.4 and 1.5 µg/kg, and 0.5 and 1.5 µg/kg, respectively. A total of 30 Chinese gelatin medicine samples were analyzed using the established method. No drug residues were found in these samples except for one Testudinis Carapacis et Plastri (1.67 µg/kg FF) and one turtle shell glue (2.55 µg/kg FF).

Ethyl carbamate regulation and genomic expression of Saccharomyces cerevisiae during mixed-culture yellow rice wine fermentation with Lactobacillus sp.

Ethyl carbamate (EC) is a potentially carcinogenic substance present in most alcoholic beverages, especially in Chinese rice wine. Consequently, much effort has been directed at suppressing EC formation during the production of these beverages, with particular attention directed at the use of urethanase, as this enzyme can directly catalyze EC degradation. Herein, we investigated the ability of three lactic acid bacteria (Oenococcus oeni, Lactobacillus brevis, and Lactobacillus plantarum) to generate urethanase during co-cultivation with Saccharomyces cerevisiae. qPCR and transcriptomic analyses revealed that 57 genes of S. cerevisiae were significantly expressed in the presence of L. brevis, which highlighted the importance of studying urethanase-promoted EC degradation for establishing a powerful technique of EC level control. The obtained results provided deep insights into the adaptive responses of S. cerevisiae to the challenging environment of mixed-culture fermentation.

Salt-tolerant Staphylococcus bacteria induce structural and nutritional alterations of salted duck egg white.

Salted duck egg white, a major by-product of salted egg yolk production, is rich in nutrients. However, its high salinity limits its application in the food industry. In the present study, three haloduric bacterium strains (C1, C2, and C3) were isolated from Jinhua ham, and strain C1 exhibited higher ratio of the transparent circle diameter to the colony diameter (HC) and gelatin liquefaction. Strain C1 was further identified as a member of the genus Staphylococcus through gene sequencing and EzTaxon-e analyses. Salted duck egg white was fermented by strain C1, and the thermal stability, microstructure, amino acid composition, and γ-aminobutyric acid of the egg white were compared with egg white without fermentation. The fermented salted duck egg white had a significantly low salinity. Meanwhile, it increased its thermal stability compared with the control through losing an endotherm at around 85°C and forming a new endotherm peak starting at 91.8°C. Additionally, free amino acids and γ-aminobutyric acid were found only in the fermented salted duck egg white. These indicated that fermentation with salt-resistant strains could alter the structure of salted duck egg white and improve its nutritional quality.

Expression of manB Gene from Escherichia coli in Lactococcus lactis and Characterization of Its Bifunctional Enzyme, Phosphomannomutase.

Phosphomannomutase (ManB) converts mannose-6-phosphate (M-6-P) to mannose-1-phosphate (M-1-P), which is a key metabolic precursor for the production of GDP-D-mannose used for production of glycoconjugates and post-translational modification of proteins. The aim of this study was to express the manB gene from Escherichia coli in Lactococcus lactis subsp. cremoris NZ9000 and to characterize the encoded enzyme. The manB gene from E. coli K12, of 1,371 bp and encoding 457 amino acids (52 kDa), was cloned and overexpressed in L. lactis NZ9000 using the nisin-controlled expression system. The enzyme was purified by Ni-NTA column chromatography and exhibited a specific activity of 5.34 units/mg, significantly higher than that of other previously reported ManB enzymes. The pH and temperature optima were 8.0 and 50°C, respectively. Interestingly, the ManB used in this study had two substrate specificity for both mannose-1-phosphate and glucose-1-phosphate, and the specific activity for glucose-1-phosphate was 3.76 units/mg showing 70% relative activity to that of mannose-1-phosphate. This is the first study on heterologous expression and characterization of ManB in lactic acid bacteria. The ManB expression system constructed in this study canbe used to synthesize rare sugars or glycoconjugates.

Effect of gallic and protocatechuic acids on the metabolism of ethyl carbamate in Chinese yellow rice wine brewing.

It was studied that gallic and protocatechuic acids played important roles in ethyl carbamate (EC) forming. Gallic and protocatechuic acids can reduce the arginine consumption through inhibiting the arginine deiminase enzyme. Therefore, they are generally added to regulate EC catabolism in the course of yellow rice wine leavening at the third day. In this work, gallic and protocatechuic acids made little influence on the growth of Saccharomyces cerevisiae. Besides, the addition of 200mg/L gallic or protocatechuic acid could prevent the transformation from urea/citrulline to EC. Gallic acid showed better inhibiting effect that the content of EC could be reduced by 91.9% at most. Furthermore, the production of amino acids and volatile flavor compounds are not markedly affected by phenolic compounds. The discoveries reveal that EC can be reduced by supplying gallic acid or protocatechuic acid while yellow rice wine leavening.

Bacterial Diversity Analysis during the Fermentation Processing of Traditional Chinese Yellow Rice Wine Revealed by 16S rDNA 454 Pyrosequencing.

Rice wine is a traditional Chinese fermented alcohol drink. Spontaneous fermentation with the use of the Chinese starter and wheat Qu lead to the growth of various microorganisms during the complete brewing process. It's of great importance to fully understand the composition of bacteria diversity in rice wine in order to improve the quality and solve safety problems. In this study, a more comprehensive bacterial description was shown with the use of bacteria diversity analysis, which enabled us to have a better understanding. Rarefaction, rank abundance, alpha Diversity, beta diversity and principal coordinates analysis simplified their complex bacteria components and provide us theoretical foundation for further investigation. It has been found bacteria diversity is more abundant at mid-term and later stage of brewing process. Bacteria community analysis reveals there is a potential safety hazard existing in the fermentation, since most of the sequence reads are assigned to Enterobacter (7900 at most) and Pantoea (7336 at most), followed by Staphylococcus (2796 at most) and Pseudomonas (1681 at most). Lactic acid bacteria are rare throughout the fermentation process which is not in accordance with other reports. This work may offer us an opportunity to investigate micro ecological fermentation system in food industry.

A novel one-step microbial transformation of betulin to betulinic acid catalysed by Cunninghamella blakesleeana.

Betulinic acid and its derivatives are potential bioactive compounds present in nature. This study investigated the biotransformation of betulin to betulinic acid by Cunninghamella blakesleeana cells. LC-MS analysis demonstrated that betulin could be transformed into at least five products from cultured C. blakesleeana cells, among which betulinic acid was the most important. The presented method provides an attractive alternative approach to chemical synthesis, because is less time-consuming and more environmentally friendly. C. blakesleeana can transform betulin into potent derivatives with high pharmacological activities.