Optimizing Akkermansia muciniphila Isolation and Cultivation: Insights into Gut Microbiota Composition and Potential Growth Promoters in a Chinese Cohort.

The study aims to analyze the composition of the gut microbiota in Chinese individuals using metagenomic sequencing technology, with a particular focus on the abundance of Akkermansia muciniphila (Akk). To improve the efficiency of Akk isolation and identification accuracy, modifications were made to the enrichment culture medium and 16S rRNA universal primers. Additionally, potential growth-promoting factors that stimulate Akk growth were explored through in vitro screening. The research results revealed that the abundance of Akk in Chinese fecal samples ranged from 0.004% to 0.4%. During optimization, a type of animal protein peptide significantly enhanced the enrichment efficiency of Akk, resulting in the isolation of three Akk strains from 14 fecal samples. Furthermore, 17 different growth-promoting factors were compared, and four factors, including galactose, sialic acid, lactose, and chitosan, were identified as significantly promoting Akk growth. Through orthogonal experiments, the optimal ratio of these four growth-promoting factors was determined to be 1:1:2:1. After adding 1.25% of this growth-promoting factor combination to the standard culture medium, Akk was cultivated at 37° for 36 h, achieving an OD600nm value of 1.169, thus realizing efficient proliferation and optimized cultivation of Akk. This study provides important clues for a deeper understanding of the gut microbiota composition in Chinese individuals, while also offering effective methods for the isolation and cultivation of Akk, laying the groundwork for its functional and application research in the human body.

Changes in glycosylated proteins in colostrum and mature milk and their implication.

Introduction: Glycosylation is one of the essential post-translational modifications that influences the function of milk proteins. Methods: In the present study, 998 proteins and 764 glycosylated sites from 402 glycoproteins were identified in human milk by TMT labeling proteomics. Compared to human milk proteins, the glycoproteins were mainly enriched in cell adhesion, proteolysis, and defense/immune process. Results: The abundance of 353 glycosylated sites and their 179 parent proteins was quantified. After normalization to their parent protein's abundance, 78 glycosylated sites in 56 glycoproteins and 10 glycosylated sites in 10 glycoproteins were significantly higher in colostrum and mature milk, respectively. These changed glycoproteins were mainly related to host defense. Intriguingly, one glycosylated site (Asp144) in IgA and two glycosylated sites (Asp38 and Asp1079) in tenascin are significantly upregulated even though their protein abundance was downregulated during lactation. Discussion: This study helps us figure out the critical glycosylated sites in proteins that might influence their biological function in an unbiased way.

Different responses of two maize cultivars to Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae infestation provide insights into their differences in resistance.

Spodoptera frugiperda (Lepidoptera: Noctuidae), a pest with an amazing appetite, damages many crops and causes great losses, especially maize. Understanding the differences in different maize cultivars' responses to S. frugiperda infestation is very important for revealing the mechanisms involved in the resistance of maize plants to S. frugiperda. In this study, a comparative analysis of two maize cultivars, the common cultivar 'ZD958' and the sweet cultivar 'JG218', was used to investigate their physico-biochemical responses to S. frugiperda infestation by a pot experiment. The results showed that the enzymatic and non-enzymatic defense responses of maize seedlings were rapidly induced by S. frugiperda. Frist, the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents of infested maize leaves were significantly increased and then decreased to the level of the control. Furthermore, compared with the control leaves, the puncture force values and the total phenolics, total flavonoids, and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one contents of infested leaves were significantly increased within a certain time. The superoxide dismutase and peroxidase activities of infested leaves were significantly increased in a certain period of time, while the catalase activities decreased significantly and then increased to the control level. The jasmonic acid (JA) levels of infested leaves were significantly improved, whereas the salicylic acid and abscisic acid levels changed less. Signaling genes associated with phytohormones and defensive substances including PAL4, CHS6, BX12, LOX1, and NCED9 were significantly induced at certain time points, especially LOX1. Most of these parameters changed greater in JG218 than in ZD958. Moreover, the larvae bioassay showed that S. frugiperda larvae weighed more on JG218 leaves than those on ZD958 leaves. These results suggested that JG218 was more susceptible to S. frugiperda than ZD958. Our findings will make it easier to develop strategies for controlling S. frugiperda for sustainable maize production and breeding of new maize cultivars with increased resistance to herbivores.

Bacterial Diversity of Sun-Dried Spanish Mackerel in Dalian and Application of Lactobacillus plantarum X23 as a Biopreservative.

ABSTRACT: Sun-dried Spanish mackerel is a common food in Dalian and made by adding salt and sun drying, which has special physical, chemical, and microbiological properties. In this study, the physicochemical properties and microbial composition of commercially available sun-dried Spanish mackerel in Dalian were assessed, and some Lactobacillus strains were screened as a biopreservative for sun-dried Spanish mackerel preparation. The results showed that the total volatile base nitrogen content in the traditional sun-dried Spanish mackerel samples from Dalian was within 30 mg/100 g, the histamine content was 7 to 17 mg/kg, and the dominant bacteria at the genus level were Lactobacillus, Psychrobacter, and Ralstonia. A strain with biopreservative potential was isolated from a sun-dried Spanish mackerel sample, identified as L. plantarum species by 16S rDNA sequencing, and assigned as L. plantarum X23. Fresh Spanish mackerel flesh was treated with 16% brine and L. plantarum X23 at a dose of 107 CFU/mL and then dried in the sun. The sun-dried Spanish mackerel flesh treated with 16% brine and L. plantarum X23 showed a decreased histamine and acid value, increased free amino acid content, and a higher sensory score compared with the sun-dried Spanish mackerel without L. plantarum X23 treatment (P < 0.05). In conclusion, the sun-dried Spanish mackerel purchased from the supermarkets in Dalian were safely edible, and L. plantarum X23 can significantly reduce the content of histamine and putrescine in self-made, low-salt, sun-dried Spanish mackerel and has potential as a biopreservative for sun-dried Spanish mackerel preparation.

Preparation and drying of water-in-oil-in-water (W/O/W) double emulsion to encapsulate soy peptides.

Soy peptide solution (40%, w/w) was successfully encapsulated in a W1/O/W2 double emulsion produced by a two-step emulsification process. Polyglycerol polyricinoleate (PGPR) was found to be an effective inner emulsifier compared to Span 60 and lecithin to produce stable W1/O primary emulsion. The primary emulsion was subsequently emulsified into an outer aqueous phase (W2) containing octenyl succinic anhydride (OSA) starch and maltodextrin. The droplet size and encapsulation efficiency of the peptide solution in W1/O/W2 emulsion were found to depend on the W1:O ratio, peptide concentration in the inner W1 phase and homogenization condition of the secondary emulsification step. The double emulsion with the highest encapsulation efficiency (>80%) was prepared by: (i) using 40% (w/w) soy peptide solution as W1 phase; (ii) controlling W1:O ratio at 3:7 (w/w) and (iii) homogenizing the emulsion at 10,000 rpm for 3 min. The freeze-dried microcapsule powder of W1/O/W2 emulsion showed higher encapsulation efficiency (>70%) compared to spray-dried one. The freeze-dried microcapsule of W1/O/W2 double emulsion developed in this study is a promising delivery matrix to encapsulate hydrophilic ingredients including peptides. Fourier-transform infrared spectroscopy (FTIR) spectra of the microcapsule powder indicated good compatibility between peptide and encapsulants.

Screening for Cholesterol-Lowering Probiotics from Lactic Acid Bacteria Isolated from Corn Silage Based on Three Hypothesized Pathways.

A total of 85 strains of lactic acid bacteria were isolated from corn silage in this study and analyzed in vitro for their cholesterol removal, NPC1L1 protein down-regulation and bile salt deconjugation ability, respectively. Nineteen strains were selected for further analysis for their probiotic potential. Finally, 3 strains showing better probiotic potential were evaluated for their cholesterol-lowering activity in hamsters. The strains showing the greater cholesterol removal and NPC1L1 protein down-regulation activity had no significant effects on serum and hepatic cholesterol levels in hamsters (p > 0.05). However, Lactobacillus plantarum CAAS 18008 (1 × 109 CFU/d) showing the greater bile salt deconjugation ability significantly reduced serum low-density lipoprotein cholesterol, total cholesterol, and hepatic total cholesterol levels by 28.8%, 21.7%, and 30.9%, respectively (p < 0.05). The cholesterol-lowering mechanism was attributed to its bile salt hydrolase activity, which enhanced daily fecal bile acid excretion levels and thereby accelerated new bile acid synthesis from cholesterol in liver. This study demonstrated that the strains showing greater cholesterol removal and NPC1L1 protein down-regulation activity in vitro hardly reveal cholesterol-lowering activity in vivo, whereas the strains showing greater bile salt deconjugation ability in vitro has large potential to decrease serum cholesterol levels in vivo.

A comparative analysis of derivatization strategies for the determination of biogenic amines in sausage and cheese by HPLC.

The six biogenic amines in sausage and cheese were analyzed by HPLC with UV detection after off-line derivatization with dansyl chloride, 9-fluorenylmethoxycarbonyl chloride, benzoyl chloride and dabsyl chloride, respectively. The results showed that both the off-line 9-fluorenylmethoxycarbonyl and dabsyl derivatization were not suitable for HPLC analysis of biogenic amines when batch injection was used because the derivatives were instable, whereas both the off-line dansyl and benzoyl derivatization were suitable for HPLC analysis of biogenic amines when batch injection was used, but the latter needed to maintain the derivatives at 4 °C to ensure that benzoylated tyramine was not degraded when waiting for the analysis. The off-line dansyl derivatization had an obvious advantage in the analysis of biogenic amines in sausage and cheese samples by HPLC combined with batch injection because the method has a wider linear range and higher sensitivity, accuracy, precision and stability of the derivatives.

Beneficial Metabolic Effects of 2',3',5'-Triacetyl-N6-(3-hydroxylaniline) adenosine in Multiple Biological Matrices and Intestinal Flora of Hyperlipidemic Hamsters.

Hyperlipidemia is one of the main causes of obesity, type 2 diabetes mellitus (T2DM), and atherosclerosis. The adenosine derivative, 2',3',5'-triacetyl-N6-(3-hydroxylaniline) adenosine (IMM-H007) is an effective lipid-lowering compound that has important implications for the development of lipid-lowering drugs. Metabolomic analysis based on 1H NMR was used to monitor dynamic changes in diverse biological media including serum, liver, urine, and feces in response to high-fat diet (HFD) and IMM-H007 treatments. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography (GC) analyses were performed to quantify the bile acids and fatty acids in the liver and feces. Fecal microbiome profiling was performed using Illumina sequencing of the 16S rRNA ( 16S rRNA) gene. IMM-H007 improved the metabolism of carbohydrate, ketone bodies, fatty acids, amino acids, and bile acids in hyperlipidemic hamsters. The correlation between metabolite changes was explored in different biological media. Significant changes in gut microbiota were observed in the HFD and IMM-H007 treatment groups. In the HFD group at the phylum level, we found high levels of the Firmicutes genus and low levels of Bacteroidetes. In contrast, the administration of IMM-H007 reversed the levels of Firmicutes and Bacteroidetes. This reversal suggested that IMM-H007 may have the ability to regulate the composition of the gut flora. We also analyzed the correlation between the gut flora and the metabolites. Our results indicate that IMM-H007 treatment improves the hyperlipidemic metabolism and the structure of the gut microbiota in hyperlipidemic hamsters.

Comparative proteomics analysis of human and ruminant milk serum reveals variation in protection and nutrition.

In present study, 198, 169, 213 and 128 proteins were identified and quantified in human, cow, goat and yak milk serum respectively by using proteomics techniques. Large variations were observed between human and ruminant milk proteins. Human milk contained higher concentration of mucosal immune response, complement proteins and regulators. The concentration of bactericidal proteins were relatively higher in ruminants milk. Human milk exclusively expressed proteins important for delivery or utilization of nutrients. Peptidase inhibitors prevent the bioactive proteins/peptides in human milk from gastral-intestinal digestion. Protein composition among ruminants milk was similar but with variations. Goat milk contained high level of complement proteins but low level of corresponding regulators. In addition, the acute phase proteins were significantly higher in goat milk. Of note, osteopontin in yak milk was enriched, which could offer an alternative source for producing osteopontin and revealed possible nutritional value of yak milk.

Identification and Functional Validation of Autolysis-Associated Genes in Lactobacillus bulgaricus ATCC BAA-365.

Lactic acid bacteria (LAB) are important organisms in food production. Indeed, LAB autolysis is very critical in dairy processing. For example, it influences the development of cheese flavor by releasing intracellular enzymes, and controls cell growth in yogurts and probiotic products. Two component systems (TCS) constitute essential environmental sensors and effectors of signal transduction in most bacteria. In the present work, mutants of one TCS (LBUL_RS00115/LBUL_RS00110) were generated to assess the relationship between TCS and cell autolysis. The mutants displayed decreased autolysis in comparison with wild type; meanwhile, complementation reversed this effect. The interaction between LBUL_RS00115 and LBUL_RS00110 was confirmed by yeast two-hybrid analysis. These observations suggested that the TCS (LBUL_RS00115/LBUL_RS00110) was involved in autolysis in Lactobacillus delbrueckii subsp. bulgaricus.

Identification of Quorum Sensing Signal Molecule of Lactobacillus delbrueckii subsp. bulgaricus.

Many bacteria in nature use quorum sensing (QS) to regulate gene expression. The quorum sensing system plays critical roles in the adaptation of bacteria to the surrounding environment. Previous studies have shown that during high-density fermentation, the autolysis of lactic acid bacteria was regulated by the QS system, and the two-component system (TCS, LBUL_RS00115/LBUL_RS00110) is involved in the autolysis of Lactobacillus delbrueckii subsp. bulgaricus. However, the QS signal molecule, which regulates this pathway, has not been identified. In this study, we compared the genome of Lactobacillus bulgaricus ATCC BAA-365 with the locus of seven lactobacillus QS systems; the position of the QS signal molecule of Lactobacillus bulgaricus ATCC BAA-365 was predicted by bioinformatics tool. Its function was identified by in vitro experiments. Construction of TCS mutant by gene knockout of LBUL_RS00115 confirmed that the signal molecule regulates the density of the flora by the TCS (LBUL_RS00115/LBUL_RS00110). This study indicated that quorum quenching and inhibition based on the signal molecule might serve as an approach to reduce the rate of autolysis of LAB and increase the number of live bacteria in fermentation.

Comparative proteomics of milk fat globule membrane in goat colostrum and mature milk.

As an important nutrient source in large area of world, the composition and nutritional value of goat milk are not well deliberated. Detailed annotation of protein composition is essential to address the physiological and nutritional value of goat milk. In the present study, 423 colostrum and mature goat milk fat globule membrane (MFGM) proteins were identified. The abundance of 189 proteins was significantly different between colostrums and mature milk MFGM. The acute phase proteins were higher in colostrums MFGM than those in mature milk MFGM which protected newborns at the beginning of life. Proteins related to synthesis and secretion were conserved through lactation to ensure the milk production. Of note, long term depression (LTD) proteins were observed in colostrum and mature milk MFGM. Milk LTD proteins could be potential biomarkers for diagnosis of lactation related depressive syndromes and should be taken into considerations of their effects on newborns.

Short communication: Antiproliferative effect of 8 different Lactobacillus strains on K562 cells.

Some strains of Lactobacillus genus have antiproliferative activities against cancer cells. However, until now, the exact effector molecules of Lactobacillus strains with anticancer activity have not been identified. The aim of the present study was to explore which fraction of the Lactobacillus cells exerts the highest antiproliferative effect. For this purpose, the heat-killed bacterial cells, bacterial cell wall extract, and genomic DNA of 8 Lactobacillus strains were prepared to assess their antiproliferative activities against human myeloid leukemia cell lines K562. The heat-killed bacterial cells of the 8 lactobacilli strains exerted antiproliferative effect on K562 cells, and the inhibition rates exerted by the heat-killed bacterial cells of the strains G15AL, M5AL, SB31AL, SB5AL, and T3AL were significantly higher than those exerted by the cell walls and genomic DNA of the strains. The bacterial DNA of G15AL exerted higher antiproliferative effect on K562 cells. The exact effector molecules and the effect mechanism of the strains should be further explored for the application of these strains as probiotic strains or bioactive probiotic molecules.

CD44(+)/CD24(-) cells are transit progenitors and do not determine the molecular subtypes and clinical parameters in breast carcinomas.

CD44(+)/CD24(-) cells have been associated with breast cancer stem/progenitor cell features. However, the status of this phenotype cells in normal, benign and malignant breast tissues has not been studied, and the clinical correlation of this subpopulation in breast cancer is not fully understood. The present study sought to identify these cells in a series of normal, benign, and malignant breast tissues and explore their correlation to the molecular subtypes of breast carcinoma and conventional pathological features. Double-staining immunohistochemistry (DIHC) of CD44 and CD24 was performed on 30 normal breast tissues, 30 breast fibroadenomas (FA), 60 breast invasive ductal carcinomas (IDC), and 3 breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-231). In the normal breast tissues and FAs, three phenotypes were observed including CD44(+)/CD24(+), CD44(+)/CD24(-), and CD44(-)/CD24(-) cells. In the IDCs, CD44(-)/CD24(+) cells were detected, in addition to the three aforementioned phenotypes. The strong positive rate (+++, incidence >60%) of CD44(+)/CD24(-) was significantly increased from normal breast tissue, FAs to IDCs (0.0%-->6.7%-->21.7%). However, the CD44(+)/CD24(-) cells didn't correlate with ages of patients, lymph node metastasis, tumor size, molecular subtypes, and the expression of ER, PR, HER-2, PS2, Bcl-2, nm23. The proportion of CD44(+)/CD24(-) cells in MCF-7, MDA-MB-468, and MDA-MB-231 was about 1, 5, and 80%, respectively. The results indicate that the CD44(+)/CD24(-) cells are transit progenitors and have no association with the molecular subtypes and clinicopathological parameters in the IDCs.

[Quantity and distribution of CD44+/CD24- cells in breast cancer tissue and the cell lines].

OBJECTIVE: To study the distribution and quantity of CD44+/CD24- cells in breast cancer tissue and the cell lines, and as well as its correlation with the expression of various breast cancer markers and molecular subtyping of breast carcinoma. METHODS: The expression of CD44/CD24, estrogen receptor, progesterone receptor, HER2, human estrogen-induced protein PS2, bcl-2 and nm23 in 60 cases of invasive ductal carcinoma of breast were studied by either single or double immunohistochemical staining. The co-expression of CD44 and CD24 in 3 breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-231) was also examined. RESULTS: The quantity and distribution of CD44+/CD24- cells varied greatly and no specific patterns were identified. The percentage of CD44+/CD24- in breast cancer was 65%. The amount of CD44+/CD24- cells did not correlate with the age of patients, lymph node metastasis, tumor size, molecular subtypes and expression of various breast cancer markers in breast carcinoma. The proportion of CD44+/CD24- cells in MCF-7, MDA-MB-468, and MDA-MB-231 cell lines was <1%, 5% and >80%, respectively. CONCLUSIONS: CD44+/CD24- cells are demonstrated in certain breast cancer tissues and cell lines. However, there is no relationship obtained between the quantity or the distribution of these cells and the molecular subtyping or the clinicopathologic parameters in breast cancer.