Antibacterial characteristics and mechanistic insights of combined tea polyphenols, Nisin, and epsilon-polylysine against feline oral pathogens: a comprehensive transcriptomic and metabolomic analysis.

AIMS: This study evaluates the antibacterial characteristics and mechanisms of combined tea polyphenols (TPs), Nisin, and ε-polylysine (PL) against Streptococcus canis, Streptococcus minor, Streptococcus mutans, and Actinomyces oris, common zoonotic pathogens in companion animals. METHODS AND RESULTS: Pathogenic strains were isolated from feline oral cavities and assessed using minimum inhibitory concentration (MIC) tests, inhibition zone assays, growth kinetics, and biofilm inhibition studies. Among single agents, PL exhibited the lowest MIC values against all four pathogens. TP showed significant resistance against S. minor, and Nisin against S. mutans. The combination treatment (Comb) of TP, Nisin, and PL in a ratio of 13:5:1 demonstrated broad-spectrum antibacterial activity, maintaining low MIC values, forming large inhibition zones, prolonging the bacterial lag phase, reducing growth rates, and inhibiting biofilm formation. RNA sequencing and metabolomic analysis indicated that TP, Nisin, and PL inhibited various membrane-bound carbohydrate-specific transferases through the phosphoenolpyruvate-dependent phosphotransferase system in S. canis, disrupting carbohydrate uptake. They also downregulated glycolysis and the citric acid cycle, inhibiting cellular energy metabolism. Additionally, they modulated the activities of peptidoglycan glycosyltransferases and d-alanyl-d-alanine carboxypeptidase, interfering with peptidoglycan cross-linking and bacterial cell wall stability. CONCLUSIONS: The Comb therapy significantly enhances antibacterial efficacy by targeting multiple bacterial pathways, offering potential applications in food and pharmaceutical antimicrobials.

Optimization of concentrations of different n-3PUFAs on antioxidant capacity in mouse hepatocytes.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), mainly including α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), possess antioxidant properties and play a crucial role in growth and development. However, the combined effects of ALA, EPA, and DHA at different concentrations have rarely been reported. This work explored the effects of EPA, ALA, and DHA on the viability and antioxidant capacity of mouse hepatocytes, with the objective of enhancing the antioxidant capacity. Within the appropriate concentration range, cell viability and the activity of glutathione S-transferase, superoxide dismutase, and catalase were increased, while the oxidation products of malondialdehyde and the level of intracellular reactive oxygen species were obviously reduced. Thus, oxidative stress was relieved, and cellular antioxidant levels were improved. Finally, response surface optimization was carried out for EPA, ALA, and DHA, and the model was established. The antioxidant capacity of the cells was highest at EPA, ALA, and DHA concentrations of 145.46, 405.05, and 551.52 µM, respectively. These findings lay the foundation for further exploration of the interactive mechanisms of n-3 PUFAs in the body, as well as their applications in nutraceutical food.

Casein phosphopeptide calcium chelation: preparation optimization, in vitro gastrointestinal simulated digestion, and peptide fragment exploration.

BACKGROUND: Calcium is important in the formation of bones and teeth, cell metabolism, and other physiological activities. In this work, casein phosphopeptide-calcium chelate (CPP-Ca) was synthesized and the optimal process parameters for the chelation reaction were obtained. The bioavailability of calcium in CPP-Ca was investigated by in vitro gastrointestinal simulated digestion. The existence of phytic acid and oxalic acid in the digestion system was evaluated to clarify the calcium holding ability of casein phosphopeptide (CPP). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify oligopeptides from CPP-Ca. RESULTS: The optimal process parameters for the chelation reaction were: peptide concentration 7.76 mgmL-1 , pH 8.54, and reaction temperature 43.3 °C. The digestion in vitro results indicated that the calcium release rate of CPP-Ca in the stomach for 2 h reached 85%, and about 50% of the ionized calcium was re-chelated with CPP in the intestine. Phytic acid and oxalic acid could lead to a sharp decrease in soluble calcium but around 50% of the calcium was still retained in the form of chelates in the presence of CPP. The LC-MS/MS identified 19 casein-derived oligopeptides after digestion, and calcium modifications were found on eight peptides derived from ß-casein and αs2 -casein. CONCLUSIONS: This study clarified the excellent calcium holding capacity of CPP in the presence of phytic acid and oxalic acid. Liquid chromatography-tandem mass spectrometry also revealed peptide changes, and identified peptides that chelate with calcium. These findings provided significant insights that could be relevant to the further utilization and product development of peptide-calcium chelate in the food industry. © 2023 Society of Chemical Industry.

In vitro gastrointestinal simulated digestion of three plant proteins: determination of digestion rate, free amino acids and peptide contents.

Cassava protein (CP), barley protein (BP) and yellow pea protein (YPP) are important nutrient and integral constituent of staple in pet foods. It is known that the digestion of proteins directly influences their absorption and utilisation. In the present work, we performed in vitro simulated gastrointestinal digestion of three plant proteins as a staple for dog and cat food. The digestion rate of CP, BP and YPP in dog food was 56.33 ± 0.90%, 48.53 ± 0.91%, and 66.96 ± 0.37%, respectively, whereas the digestion rate of CP, BP, and YPP in cat food was 66.25 ± 0.72%, 43.42 ± 0.83%, and 58.05 ± 0.85%, respectively. Using SDS-polyacrylamide gel electrophoresis to determine the molecular weight (MW) of each protein and the products of their digestion, it was revealed that MW of digestion samples decreased, and MW during the small intestine phase was lower than that during the gastric phase. Peptide sequences of digested products were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and it was found that the total number of peptides in the small intestine digestion samples was higher than that in the gastric phase samples. The MW of peptides obtained from CP was within the range of 1000-1500 Da, while MW of peptides derived from BP and YPP was within the range of 400-2000 Da. In addition, free amino acids were mainly produced in the small intestine phase. Furthermore, the percentage of essential amino acids in the small intestine phase (63 ~ 82%) was higher than that in the gastric phase (37 ~ 63%). Taken together, these findings contribute to the current understanding of the utilisation of plant proteins in dog and cat foods and provide important insights into the selection and application of plant proteins as a staple in dog and cat foods.

Transcriptomic and metabolomic analysis of prebiotics utilization by Bifidobacterium animalis.

In this study, the utilization mechanism of oligosaccharides by Bifidobacterium was investigated through the transcriptome sequencing and non-targeted metabolomics technology of Bifidobacterium animalis cultured with fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS). The results showed that FOS affected the synthesis of adenosine triphosphate binding transporters (ABC transporters) by increasing the expression levels of msmE, msmG, and gluA. Similarly, GOS improved aminoacyl-tRNA synthases by upregulating the expression of tRNA-Ala, tRNA-Pro, and tRNA-Met. Bifidobacterium animalis cultured with FOS and GOS produced different metabolites, such as histamine, tartaric acid, and norepinephrine, with the functions of inhibiting inflammation, alleviating depression and diseases related to brain and nervous system and maintaining body health. Furthermore, the transcriptome and metabolome analysis results revealed that FOS and GOS promoted the growth and metabolism of Bifidobacterium animalis by regulating the related pathways of carbohydrate, energy, and amino acid metabolism. Overall, the experimental results provided significant insights into the prebiotic effects of FOS and GOS.

Variations in gut microbiome and metabolites of dogs with acute diarrhea in poodles and Labrador retrievers.

For different breeds of dogs with acute diarrhea, the gut microbiota and metabolome profiles are unclear. This prospective observational study analyzed the gut microbiomes of poodles with acute diarrhea and Labrador retrievers with acute diarrhea based on 16S amplicon sequencing, with respective healthy dogs as controls. Fecal non-target metabolomics and metagenomics were performed on poodles with acute diarrhea. This study found that the diversity and structure of the microbial community differed significantly between the two breeds in cohorts of healthy dogs. Two breeds of dogs with acute diarrhea demonstrated different changes in microbial communities and metabolic functions. The metabolism of starch and sucrose was significantly decreased in dogs with acute diarrhea, which may be attributed to the reduced activity of dextran dextrinase. Non-targeted metabolomics identified 21 abnormal metabolic pathways exhibited by dogs with acute diarrhea, including starch, amino acid, bile acid metabolism, etc., and were closely related to specific intestinal flora. This study provided new insights into breed specificity and the development of dietary treatment strategy in canine gastrointestinal disease.

An electrochemical aptasensor based on cocoon-like DNA nanostructure signal amplification for the detection of Escherichia coli O157:H7.

We developed an electrochemical aptasensor based on cocoon-like DNA nanostructures as signal tags for highly sensitive and selective detection of Escherichia coli O157:H7. The stable cocoon-like DNA nanostructures synthesized by the rolling circle amplification reaction were loaded with hemin as electrochemical signal tags to amplify the signals. The single-stranded DNA capture probes were modified on the surface of a Au electrode via a Au-S bond. The E. coli O157:H7 specific aptamer and capture probe formed double-stranded DNA structures on the Au electrode. The aptamer preferentially bound to E. coli O157:H7, causing the dissociation of some aptamer-capture probes and releasing some capture probes. Subsequently, the free capture probes hybridized with the DNA nanostructures through the cDNA sequence. Under optimal conditions, the change in the electrochemical signal was proportional to the logarithm of E. coli O157:H7 concentration, from 10 to 106 CFU mL-1, and the detection limit was estimated to be 10 CFU mL-1. The electrochemical aptasensor could be readily used to detect various pathogenic bacteria and to provide a new method of early diagnosis of pathogenic microorganisms.

A sensitive biosensor for determination of pathogenic bacteria using aldehyde dehydrogenase signaling system.

Aldehyde dehydrogenase (ALDH) was first developed as an enzymatic signaling system of a biosensor for sensitive point-of-care detection of pathogenic bacteria. ALDH and specific aptamers to Salmonella typhimurium (S. typhimurium), as organic components, were embedded in organic-inorganic nanocomposites as a biosensor signal label, integrating the functions of signal amplification and target recognition. The biosensing mechanism is based on the fact that ALDH can catalyze rapid oxidation of acetaldehyde into acetic acid, resulting in pH change with portable pH meter readout. The altered pH exhibited a linear relationship with the logarithm of S. typhimurium from 102 to 108 CFU/mL and detection limit of 46 CFU/mL. Thus, the proposed biosensor has potential application in the diagnosis of pathogenic bacteria.

Sandwich immunoassay based on antimicrobial peptide-mediated nanocomposite pair for determination of Escherichia coli O157:H7 using personal glucose meter as readout.

A sandwich immunoassay was developed for determination of E. coli O157:H7. This is based on an antimicrobial peptide-mediated nanocomposite pair and uses a personal glucose meter as signal readout. The antimicrobial peptides, magainins I, and cecropin P1 were employed as recognition molecules for the nanocomposite pair, respectively. With a one-step process, copper phosphate nanocomposites embedded by magainins I and Fe3O4 were used as "capturing" probes for bacterial magnetic isolation, and calcium phosphate nanocomplexes composed of cecropin P1 and invertase were used as signal tags. After magnetic separation, the invertase of the signal tags hydrolyzed sucrose to glucose, thereby converting E. coli O157:H7 levels to glucose levels. This latter can be quantified by a personal glucose meter. Under optimal conditions, the concentration of E. coli O157:H7 can be determined in a linear range of 10 to 107 CFU·mL-1 with a detection limit of 10 CFU·mL-1. The method was successfully applied to the determination of E. coli O157:H7 in milk samples. Graphical abstract Schematic representation of sandwich immunoassay for E. coli O157:H7. One-pot synthetic of Fe3O4-magainins I nanocomposites (MMP) were used for magnetic capture. Cecropin P1-invertase nanocomposites (PIP) were used as signal tags. A personal glucose meter was used as readout to determine the target.

Immunoassay for pathogenic bacteria using platinum nanoparticles and a hand-held hydrogen detector as transducer. Application to the detection of Escherichia coli O157:H7.

An innovative approach is presented for portable and sensitive detection of pathogenic bacteria. A novel synthetic hybrid nanocomposite encapsulating platinum nanoparticles, as a highly efficient catalyst, catalyzes the hydrolysis of the ammonia-borane complex to generate hydrogen gas. The nanocomposites are used as a label for immunoassays. A portable hand-held hydrogen detector combined with nanocomposite-induced signal conversion was applied for point-of-care testing of pathogenic bacteria. A hand-held hydrogen detector was used as the transducer. Escherichia coli O157:H7 (E. coli O157: H7), as detection target, formed a sandwich structure with magnetic beads and hybrid nanocomposites. Magnetic beads were used for separation of the sandwich structure, and hybrid nanocomposites as catalysts to catalyze the generation of hydrogen from ammonia-borane. The generated hydrogen was detected by a hydrogen detector using an electrochemical method. E. coli O157:H7 has a detection limit of 10 CFU·mL-1. The immunosensor made the hand-held hydrogen detector a point-of-care meter to be used outdoors for the detection and quantification of targets beyond hydrogen. Graphical abstract Schematic presentation of one-pot synthetic peptide-Cu3(PO4)2 hybrid nanocomposites embedded PtNPs (PPNs), encapsulating many Pt particles. The PPNs acts as an ideal immunoprobe for hand-held H2 detector signal readouts, by transforming pathogenic bacteria recognition events into H2 signals.

Structural changes and in vitro bioaccessibility of CPP-febisgly complexes: Dependence on iron load.

The effect of casein phosphopeptides (CPP) and ferrous bisglycinate (FebisGly) at different ratios (1:20, 1:10, and 1:5 w/w) on iron supplementation was investigated. The in vitro bioaccessibility, structural changes, antioxidant activity, and the effect of absorption inhibitors were also explored. The results demonstrated that CPP enhanced the bioaccessibility of FebisGly by 68.72 % ± 0.18 % and increased the ß-sheet content from 21.60 % ± 0.23 % to 67.92 % ± 0.12 %, forming a stable secondary structure. The particle size distribution (PSD) and rheological analyses indicated that CPP significantly contributed to the formation of chelated irons, resulting in a uniform PSD and enhanced viscoelasticity. Moreover, it prolonged the gastric emptying time, reducing gastric irritation further. The carboxyl and amino groups in the CPP molecules participated in chelation reaction, improved the antioxidant activity, and competed with phytic acid, tannic acid, and cellulose for iron. Overall, these results laid a foundation for developing novel iron supplementation strategies.

Metabolic reprogramming of corn oligopeptide in regulating sodium nitrite-induced canine hepatocyte injury via TGF/NF-κB signaling pathways and aminoacyl-tRNA biosynthesis.

Sodium nitrite (SN), a prevalent food preservative, is known to precipitate hepatotoxicity upon exposure. This study elucidates the hepatoprotective effects of corn oligopeptide (COP) and vitamin E (VE) against SN-induced hepatic injury in canine hepatocytes. Canine liver cells were subjected to SN to induce hepatotoxicity, followed by treatment with COP and VE. Evaluations included assays for cell viability, oxidative stress markers, apoptosis, and inflammatory cytokines. Additionally, transcriptomic and metabolomic analyses were performed to delineate the underlying molecular mechanisms. The findings demonstrated that COP and VE significantly ameliorated SN-induced cytotoxicity, oxidative stress, and apoptosis. It was evidenced by restored cell viability, enhanced antioxidant enzyme activity, reduced cytoplasmic enzyme leakage, and decreased levels of malondialdehyde and inflammatory cytokines, with COP showing superior efficacy. The RNA sequencing revealed that COP treatment suppressed the SN-activated aminoacyl-tRNA biosynthesis pathway and TGF-ß/NF-κB signaling pathways, thereby mitigating amino acid depletion, apoptosis, and inflammation. Moreover, COP treatment upregulated genes associated with protein folding, bile acid synthesis, and DNA repair. Metabolomic analysis corroborated these results, showing that COP restored amino acid levels and enhanced bile acid metabolism, alleviating SN-induced metabolic disruptions. These findings offered significant insights into the protective mechanisms of COP underscoring its prospective application in treating liver injuries.

The influence and mechanism of fish collagen peptide and egg yolk lecithin on proliferation and lipid composition in feline adipocytes.

The influences of fish collagen peptide (FCP) and egg yolk lecithin (EYL) on the proliferation, fat accumulation and triglyceride content in feline adipocytes were investigated in this work, aiming at unveiling the mechanism of fat accumulation for cheek of feline animals. The lipogenic changes of adipocytes in the presence of FCP and EYL were determined by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The results demonstrated that FCP of 10 mg/mL had the strongest cell activity, with a relative increment rate of 156 ± 0.23%, and the triglyceride content reached 215.9 ± 3.86 mmol/L. By comparison, it was observed that an EYL concentration of 5 mg/mL elicited the highest cell activity, exhibiting a relative increment rate of 152 ± 0.60%, and the level of triglyceride content was noted to reached 256.56 ± 25.68 mmol/L. After the feline adipocytes were treated with different concentrations of two active substances, fat formation and lipid droplets were found by oil red O staining. Liposome analyses confirmed that the formation of lipid compounds was regulated by FCP and EYL through pathways involved in lipid metabolism, notably including inositol phosphate insulin resistance, and phosphatidylinositol signaling pathways. This regulation was found to enhance cell vitality and facilitate fat accumulation. These findings provide a new strategy for the development of nutritional and healthy products or foods that promote feline cheek.

Polyunsaturated fatty acids, vitamin E and lycopene alleviate ambient particulate matter organic extracts-induced oxidative stress in canine lung cells via the Nrf2/HO-1 pathway.

Exposure to environmental particulate matter (PM) causes lung damage in humans, but it is not sufficiently studied in companion animals. In this work, we found that organic extracts (OE) of PM induced oxidative stress and were more cytotoxic than water-soluble extracts (WE) of PM in canine (Canis familiaris) pulmonary alveolar type II epithelial (PAE) cells. Subsequently, we evaluated the alleviating effects of polyunsaturated fatty acid mixtures (eicosapentaenoic and docosahexaenoic acids), vitamin E, and lycopene on OE-exposed PAE cells. The results indicated that the three nutrients attenuated OE-induced oxidative stress. Compared with OE-exposed PAE cells, cells pretreated with the three nutrients exhibited a 1.7 ~ 2.2-fold reduction in reactive oxygen species, 15.58% ~ 19.96% increase in cell viability, 26.19% ~ 29.32% reduction in lactate dehydrogenase release and 33.87% ~ 40.10% reduction in intracellular malondialdehyde. Meanwhile, the activities of superoxide dismutase, catalase, and glutathione peroxidase increased by 35.22% ~ 47.70%, 45.36% ~ 64.13%, and 48.56% ~ 68.18%. Besides, microscopic observation revealed that nutrients improved cell morphology, as evidenced by reduced cell shrinkage and increased apposition. Finally, the mechanisms of OE toxicity and antioxidant nutrients were explored respectively. The results revealed that OE triggered cytotoxicity by directly disrupting antioxidant enzyme activity through activation of ROS, while nutrients inhibited OE-induced cellular oxidative stress via upregulation of the Nrf2/HO-1 signaling pathway. The present study may provide significant insights into the prevention of PM-induced lung diseases by antioxidant supplementation in animals.

Casein phosphopeptide-calcium chelate: Preparation, calcium holding capacity and simulated digestion in vitro.

In this work, CPP-Ca chelate was synthesized by chelating casein phosphopeptide (CPP) and calcium and characterized by Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) Energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). The antioxidant activity and calcium holding capacity of CPP-Ca were evaluated and its secondary structure transition was monitored during gastrointestinal digestion by in situ Raman spectroscopy. The results demonstrated that calcium chelating rate reached 40 % and calcium ion was bound to CPP mainly through the interaction of carboxyl and amino groups. The result of calcium holding capacity confirmed the formation of calcium phosphate precipitates could be delayed by 10-15 min with increasing CPP concentration. In vitro simulated digestion revealed CPP-Ca exhibited excellent calcium solubility and its secondary structural changes occurred, especially α-helix and ß-sheet content. These findings provided significant insights into enhancing bioavailability of calcium supplements and developing of calcium functional foods for human and animals.

Identification of Gut Microbiome and Metabolites Associated with Acute Diarrhea in Cats.

Changes in diet and environment can lead to acute diarrhea in companion animals, but the composition and interactions of the gut microbiome during acute diarrhea remain unclear. In this multicenter case-control study, we investigated the relationship between intestinal flora and acute diarrhea in two breeds of cats. Acutely diarrheic American Shorthair (MD, n = 12) and British Shorthair (BD, n = 12) and healthy American Shorthair (MH, n = 12) and British Shorthair (BH, n = 12) cats were recruited. Gut microbial 16S rRNA sequencing, metagenomic sequencing, and untargeted metabolomic analysis were performed. We observed significant differences in beta-diversity (Adonis, P < 0.05) across breeds and disease state cohorts. Profound differences in gut microbial structure and function were found between the two cat breeds. In comparison to healthy British Shorthair cats, Prevotella, Providencia, and Sutterella were enriched while Blautia, Peptoclostridium, and Tyzzerella were reduced in American Shorthair cats. In the case-control cohort, cats with acute diarrhea exhibited an increased abundance of Bacteroidota, Prevotella, and Prevotella copri and a decreased abundance of Bacilli, Erysipelotrichales, and Erysipelatoclostridiaceae (both MD and BD cats, P < 0.05). Metabolomic analysis identified significant changes in the BD intestine, affecting 45 metabolic pathways. Moreover, using a random forest classifier, we successfully predicted the occurrence of acute diarrhea with an area under the curve of 0.95. Our findings indicate a distinct gut microbiome profile that is associated with the presence of acute diarrhea in cats. However, further investigations using larger cohorts of cats with diverse conditions are required to validate and extend these findings. IMPORTANCE Acute diarrhea is common in cats, and our understanding of the gut microbiome variations across breeds and disease states remains unclear. We investigated the gut microbiome of two cat breeds (British Shorthair and American Shorthair) with acute diarrhea. Our study revealed significant effects of breeds and disease states on the structure and function of the gut microbiota in cats. These findings emphasize the need to consider breed-related factors in animal nutrition and research models. Additionally, we observed an altered gut metabolome in cats with acute diarrhea, closely linked to changes in bacterial genera. We identified a panel of microbial biomarkers with high diagnostic accuracy for feline acute diarrhea. These findings provide novel insights into the diagnosis, classification, and treatment of feline gastrointestinal diseases.

Ambient particulate matter (PM10)-induced injury in feline lung cells and nutritional intervention.

Ambient particulate matter (APM) is extremely harmful to life's health. In this study, we investigated cellular injury in cat (Felix catus) lung cells (FCA-L2) exposed to organic and water-soluble extracts from APM. As well, the protective effect of vitamin E (VE), lycopene and a mixture of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (molar concentration ratio of 2:1) against this damage was evaluated. Organic and water-soluble extracts induced oxidative stress in FCA-L2 cells, as evidenced by excess reactive oxygen species production and mitochondrial damage, while treatment with VE, lycopene and EPA: DHA remarkably alleviated these indices. It was further found that treatment with EPA: DHA decreased lactate dehydrogenase and malondialdehyde, as well as increased activities of superoxide dismutase, glutathione peroxidase and catalase. Our study confirmed that nutrients mediates APM-induced oxidative stress via antioxidant proteins. Also, these findings could provide new insights into reducing APM-induced cytotoxicity by nutritional supplementation based on antioxidant compounds for animals.