Research progress on pharmacological properties and application of probiotics in the fermentation of Scutellaria baicalensis Georgi.

Scutellaria baicalensis Georgi is a medicinal herb with a rich history of use in traditional Chinese medicine. This review concentrates on the chemical constituents of Scutellaria baicalensis Georgi, with a particular emphasis on flavonoids such as baicalin, baicalein, and wogonin. Additionally, it examines the effects of probiotic fermentation on the plant's chemical profile and pharmacological actions. Evidence suggests that probiotic fermentation markedly modifies the bioactive components of Scutellaria baicalensis Georgi, thereby augmenting its medicinal potency. The paper delves into the mechanisms by which the primary active constituents of Scutellaria baicalensis Georgi are altered during fermentation and how these changes influence its pharmacological properties. This review aims to lay a theoretical groundwork for the clinical utilization of Scutellaria baicalensis Georgi and the formulation of innovative therapeutic approaches.

Multiple strategies to improve extracellular secretion and activity of feruloyl esterase.

Feruloyl esterase has a wide range of applications, but there are still problems with low enzyme yield and activity, and complex purification steps. Our previous research found Lactobacillus amylovorus feruloyl esterase could be secreted extracellular in Escherichia coli. In this study, multiple strategies were implemented to maximize the extracellular production of feruloyl esterase with improved activity in E. coli. Firstly, codon-optimized feruloyl esterase was obtained based on the preference of E. coli, resulting in 41.97 % increase in extracellular secretion. Furthermore, by cascading T7 promoters, replacing the 5' UTR, randomly mutating the N-terminal sequence, and co-expressing secretory cofactors, the extracellular secretion was increased by 36.46 %, 31.25 %, 20.66 % and 25.75 %, respectively. Moreover, the feruloyl esterase were mutated to improve the substrate affinity and activity. The catalytic efficiency of Fae-Q134T and Fae-Q198A increased by 4.62-fold and 5.42-fold. Combining above strategies, extracellular feruloyl esterase activity was increased from 2013.70 U/L to 10,349.04 U/L. These results indicated that the activity and yield of feruloyl esterase secreted by E. coli were significantly increased, which laid a foundation for its industrial application.

Efficient secretion of an enzyme cocktail in Escherichia coli for hemicellulose degradation.

The use of host to secrete several hemicellulase is a cost-effective way for hemicellulose degradation. In this study, the xylose utilization gene xylAB of Escherichia coli BL21 was knocked out, and the xylanase (N20Xyl), ß-xylosidase (Xys), and feruloyl esterase (FaeLam) were co-expressed in this strain. By measuring the content of reducing sugars generated by enzymatic hydrolysis of wheat bran in the fermentation supernatant, the order of the three enzymes was screened to obtain the optimal recombinant strain of E. coli BL21/∆xylAB/pDIII-2. Subsequently, fermentation conditions including culture medium, inducer concentration, induction timing, metal ions, and glycine concentration were optimized. Then, different concentrations of wheat bran and xylan were added to the fermentation medium for degradation. The results showed that the extracellular reducing sugars content reached the highest value of 33.70 ± 0.46 g/L when 50 g/L xylan was added. Besides, the scavenging rates of hydroxyl radical by the fermentation supernatant was 81.0 ± 1.41 %, and the total antioxidant capacity reached 2.289 ± 0.55. Furthermore, it showed the growth promotion effect on different lactic acid bacteria. These results provided a basis for constructing E. coli strain to efficiently degrade hemicellulose, and the strain obtained has great potential application to transform hemicellulose into fermentable carbon source.

Effect of sugar transporter on galactose utilization in Streptococcus thermophilus.

Introduction: Streptococcus thermophilus is a traditional starter for dairy products. The lactose rich in milk is the main carbon source for the growth of S. thermophilus. However, the utilization of galactose by S. thermophilus is strain-specific, and many genetic factors can affect the sugar utilization phenotype of S. thermophilus strains. Methods: In this study, S. thermophilus A25, which is capable of utilizing lactose and galactose, was used as the starting strain to construct lactose permease-deficient mutant S. thermophilus ΔlacS. Subsequently, the complement vectors expressing complete lactose permease of S. thermophilus and its N-terminal 1-486 amino acid residues were constructed and transformed into S. thermophilus ΔlacS, respectively. Meanwhile, complement vectors expressing lactose permease and galactose/proton symporter of Escherichia coli were also constructed. Results and Discussion: Results showed that S. thermophilus ΔlacS lost the ability to utilize lactose and galactose. By measuring the growth of the recombinant strains, it was found that the strain expressing complete lactose permease of S. thermophilus recovered the growth ability in lactose and galactose medium, while the strain expressing N-terminal of lactose permease recovered the growth ability only in lactose medium. Furthermore, the transformation of S. thermophilus ΔlacS was not successful with the complement vector expressing E. coli lactose permease, while the strain expressing E. coli galactose/proton symporter could recover its growth ability in the galactose medium. These results suggest that the properties of sugar transporters play an important role in galactose utilization by S. thermophilus.

Applications of Probiotic Constituents in Cosmetics.

Over the past few decades, research on the benefits of beneficial microorganisms on skin health has expanded and attracted a lot of attention. Today, a wide range of probiotic products are becoming available. With their extensive component profiles and varied physiological effects, probiotics, as well as extracts of them, have a significant impact on cosmetics. However, the present boom in consumer interest in alternatives has broadened the probiotic industry's research and development frontiers. Considering the foregoing, it should come as no surprise that probiotics are highly valued for their proven anti-aging, skin whitening, anti-inflammatory, and photoprotective effects. This review aims to compile information on probiotics' properties, their extracts, and preparations used in cosmetics. It also further summarizes research and applications on probiotic fermentation to promote the use of probiotic fermentation products in cosmetics. Notably, this review also adds information on particular properties and mechanisms of action of probiotics, which fills a gap in the research and application of probiotics in skin treatment and care. Their antioxidant and anti-aging qualities have received particular consideration. This review provides a new basis for the broad application of probiotics in cosmetics.

Advances in Genetic Tools and Their Application in Streptococcus thermophilus.

Streptococcus thermophilus is a traditional starter. Nowadays, key aspects of S. thermophilus physiology have been revealed concerning the phenotypic traits relevant for industrial applications, including sugar metabolism, protein hydrolysis, and the production of important metabolites that affect the sensory properties of fermented foods as well as the original cooperation with Lactobacillus delbrueckii subsp. bulgaricus. Moreover, significant advances have been made in the synthetic biology toolbox of S. thermophilus based on technological advances in the genome and its sequencing and synthesis. In this review, we discuss the recently developed toolbox for S. thermophilus, including gene expression toolsets (promoters, terminators, plasmids, etc.) and genome editing tools. It can be used for both functionalized foods and therapeutic molecules for consumers. The availability of new molecular tools, including the genome editing toolbox, has facilitated the engineering of physiological studies of S. thermophilus and the generation of strains with improved technical and functional characteristics.

Recent Advances and Future Prospects of Mycosporine-like Amino Acids.

Mycosporine-like amino acids (MAAs) are a class of water-soluble active substances produced by various aquatic organisms. However, due to the limitations of low accumulation of MAAs in organisms, the cumbersome extraction process, difficult identification, and high cost, MAAs have not yet been widely used in human life. Recently, there has been an emergence of heterologous synthesis for MAAs, making increasing yield the key to the quantification and application of MAAs. This review summarizes the latest research progress of MAAs, including: (1) introducing the biodistribution of MAAs and the content differences among different species to provide a reference for the selection of research subjects; (2) elaborating the species and molecular information of MAAs; (3) dissecting the synthesis mechanism and sorting out the synthesis pathways of various MAAs; (4) summarizing the methods of extraction and identification, summarizing the advantages and disadvantages, and providing a reference for the optimization of extraction protocols; (5) examining the heterologous synthesis method; and (6) summarizing the physiological functions of MAAs. This paper comprehensively updates the latest research status of MAAs and the various problems that need to be addressed, especially emphasizing the potential advantages of heterologous synthesis in the future production of MAAs.

Probiotics: functional food ingredients with the potential to reduce hypertension.

Hypertension is an increasingly pressing public health concern across the globe. It can be triggered by a variety of factors such as age and diet, as well as the stress of modern life. The traditional treatment of hypertension includes calcium ion blockers, angiotensin II receptor inhibitors and ß-receptor blockers, but these drugs have at least some side effects. Recent studies have revealed that intestinal flora plays a vital role in maintaining and promoting human health. This is due to the type and amount of probiotics present in the flora. Probiotics can reduce hypertension symptoms through four mechanisms: regulating vascular oxidative stress, producing short-chain fatty acids, restoring endothelial cell function, and reducing inflammation. It has been reported that certain functional foods, using probiotics as their raw material, can modify the composition of intestinal flora, thus regulating hypertension symptoms. Consequently, utilizing the probiotic function of probiotics in conjunction with the properties of functional foods to treat hypertension is a novel, side-effect-free treatment method. This study seeks to summarize the various factors that contribute to hypertension, the mechanism of probiotics in mitigating hypertension, and the fermented functional foods with probiotic strains, in order to provide a basis for the development of functional foods which utilize probiotics as their raw material and may have the potential to reduce hypertension.

Platycodon grandiflorum root fermentation broth reduces inflammation in a mouse IBD model through the AMPK/NF-κB/NLRP3 pathway.

The effect of Platycodon grandiflorum (PG) on colitis and its underlying mechanism were rarely studied. In this study, Lactobacillus rhamnosus 217-1 was used to ferment PG roots, and the concentrations of platycodin-D, flavonoids, and polyphenols and the DPPH free radical scavenging rate were significantly increased. Treatment with a PG root fermentation broth (PGRFB) could reduce dextran sulfate sodium (DSS) induced ulcerative colitis (UC) in mice. Meanwhile, the PGRFB significantly reduced the content of inflammatory factors in mouse serum and the expression of inflammatory factor mRNA in the intestinal tract, regulated the polarization of M1/M2 macrophages, and increased the expression of tight junction protein mRNA in intestinal epithelial cells. In summary, it was proved that the PGRFB could inhibit the nuclear factor kappa B (NF-κB) signaling pathway and the expression of Nod-like receptor protein 3 (NLRP3) inflammasomes by activating AMP-activated protein kinase (AMPK) and lowering the release of pro-inflammatory cytokines.

Effect of the Fermentation Broth of the Mixture of Pueraria lobata, Lonicera japonica, and Crataegus pinnatifida by Lactobacillus rhamnosus 217-1 on Liver Health and Intestinal Flora in Mice With Alcoholic Liver Disease Induced by Liquor.

In this work, we discovered a new fermentation broth that can prevent and regulate alcoholic liver disease (ALD) and intestinal flora, which fermented the mixture of Pueraria lobata, Lonicera japonica, and Crataegus pinnatifida by Lactobacillus rhamnosus 217-1. The contents of polyphenols, puerarin, total isoflavones, and amino acids were significantly increased. Animal experiments showed that the fermentation broth could improve the liver indexes of ALD mice model, increase the activity of superoxide dismutase and glutathione in liver tissue, and reduce the level of malondialdehyde (MDA). Furthermore, the fermentation broth can reduce the levels of serum lipopolysaccharide (LPS), inflammatory factors interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Importantly, intestinal flora analysis showed that the fermentation broth could increase the abundance of Lactobacillales and reduce the production of Gram-negative bacteria, thereby reducing the abnormal increase in bacterial diversity caused by alcohol. In conclusion, we may have discovered a new functional food raw material with great application potential. The above findings indicate that the fermentation broth can actively regulate the intestinal flora and improve liver inflammation. The underlying mechanism might be that the fermentation broth could enhance intestinal permeability and reduce the inflammatory signals and LPS transmitted through the gut-liver axis, thereby reducing the oxidative stress and inflammation of the liver caused by alcohol.

The N-terminus of Lactobacillus amylovorus feruloyl esterase plays an important role in its secretion by Lactobacillus plantarum and Escherichia coli.

BACKGROUND: Feruloyl esterase is a multifunctional esterase with potential industrial applications. In the present study, we found the Lactobacillus amylovorus feruloyl esterase (FaeLam) could be secreted by L. plantarum and Escherichia coli. However, no signal peptide was detected in this protein as predicted by SignalP-5.0. Therefore, experiments were carried out to propose an explanation for the extracellular release of FaeLam. RESULTS: Here, we identified that the FaeLam could be secreted to the culture medium of L. plantarum CGMCC6888 and E. coli DH5α, respectively. To exclude the possibility that FaeLam secretion was caused by its hydrolytic activity on the cell membrane, the inactive FaeLamS106A was constructed and it could still be secreted out of L. plantarum and E. coli cells. Furthermore, the truncated version of the FaeLam without the N-terminal residues was constructed and demonstrated the importance of the 20 amino acids of N-terminus (N20) on FaeLam secretion. In addition, fusion of heterologous proteins with N20 or FaeLam could carry the target protein out of the cells. These results indicated the N-terminus of FaeLam played the key role in the export process. CONCLUSIONS: We proved the N-terminus of L. amylovorus FaeLam plays an important role in its secretion by L. plantarum and E. coli. To our best knowledge, this is the first reported protein which can be secreted out of the cells of both Gram-positive and Gram-negative bacteria. Furthermore, the results of this study may provide a new method for protein secretion in L. plantarum and E. coli through fusion the target protein to N20 of FaeLam.

Comparison of Enzyme Secretion and Ferulic Acid Production by Escherichia coli Expressing Different Lactobacillus Feruloyl Esterases.

Construction of recombinant Escherichia coli strains carrying feruloyl esterase genes for secretory expression offers an attractive way to facilitate enzyme purification and one-step production of ferulic acid from agricultural waste. A total of 10 feruloyl esterases derived from nine Lactobacillus species were expressed in E. coli BL21 (DE3) to investigate their secretion and ferulic acid production. Extracellular activity determination showed all these Lactobacillus feruloyl esterases could be secreted out of E. coli cells. However, protein analysis indicated that they could be classified as three types. The first type presented a low secretion level, including feruloyl esterases derived from Lactobacillus acidophilus and Lactobacillus johnsonii. The second type showed a high secretion level, including feruloyl esterases derived from Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus helveticus. The third type also behaved a high secretion level but easy degradation, including feruloyl esterases derived from Lactobacillus farciminis, Lactobacillus fermentum, and Lactobacillus reuteri. Moreover, these recombinant E. coli strains could directly release ferulic acid from agricultural waste. The highest yield was 140 µg on the basis of 0.1 g de-starched wheat bran by using E. coli expressed L. amylovorus feruloyl esterase. These results provided a solid basis for the production of feruloyl esterase and ferulic acid.

Extracellular secretion of feruloyl esterase derived from Lactobacillus crispatus in Escherichia coli and its application for ferulic acid production.

A feruloyl esterase producing strain was isolated and identified as Lactobacillus crispatus S524. Its putative feruloyl esterase was heterogeneously expressed in Escherichia coli BL21 (DE3). Interestingly, the feruloyl esterase (FaeLcr) could be secreted into the culture medium with a relative high purity of 201.7 mg/L. FaeLcr was purified from the cell-free culture supernatant and appeared as a single protein band with the molecular mass of 28 kDa by SDS-PAGE. The optimal temperature and pH were determined as 65 °C and 7.0, and it showed prominent thermo-stability and alkali-stability. Furthermore, the purified FarLcr could release a maximal amount of 199 µg ferulic acid from 0.2 g de-starched wheat bran. Meanwhile, when cultured this recombinant E. coli strain in medium supplemented with 2 g de-starched wheat bran, 1.86 mg ferulic acid was also detected. These results suggested that the recombinant strain has a great potential application in feruloyl esterase and ferulic acid production.

Endoglucanase improve the growth of homofermentative Lactobacillus spp. in ensilages.

Endoglucanase, an important component of cellulases, is used as additives in ensiling of forage crops. However, its detailed role is unclear in ensilages. In the present study, two endoglucanases Cel5 and Cel9 produced by strain Paenibacillus panacisoli SDMCC050309, previously isolated from ensiled corn stover, were identified in the cultures by microcrystalline cellulose absorption coupled with zymogram analysis. After heterologously expressed in Escherichia coli DE3 and purified, these two proteins were biochemically characterized. Cel5 was 61 kDa and showed maximal activity at pH 7.0 and 45 °C, while the maximum activity was at pH 8.0 and 65 °C for Cel9 with 97 kDa in size. Both of them could degrade carboxymethyl cellulose into cellooligosaccharides, in which cellobiose and cellotriose could be used as substrates for the growth of homofermentative strains Lactobacillus plantarum CGMCC6888 and L. farciminis CCTCC AB2016237, but not for the heterofermentative strains L. brevis SDMCC050297 and L. parafarraginis SDMCC050300. Therefore, we concluded that the added endoglucanase contributed to enhance the growth of homofermentative lactic acid bacteria for high level of lactic acid production in ensilages.

Paenibacillus panacisoli enhances growth of Lactobacillus spp. by producing xylooligosaccharides in corn stover ensilages.

The knowledge about the association of lignocellulosic biomass-degrading microbes with lactic acid bacteria (LAB) in ensilages is still limited. Paenibacillus strains are important microbes in sustainable agriculture. Here, P. panacisoli SDMCC050309 was isolated from ensiled corn stover and used as an example to investigate the effects on LAB. This strain produced at least 7 xylanases, and two of them were purified and characterized. Temperature and pH optima were determined to be 55 °C and 8.0 for Xyn10 and 40 °C and 7.0 for Xyn11, respectively. They could degraded larch wood xylan and alkali-pretreated corn stover into xylooligosaccharides (XOS). Using the produced XOS to culture Lactobacillus brevis SDMCC050297 and L. parafarraginis SDMCC050300, both of them grew well with high level of acetic acid production. The same phenomenon was observed when co-culturing those two Lactobacillus strains with P. panacisoli SDMCC050309. Therefore, P. panacisoli enhances growth of LAB by producing XOS in corn stover ensilages.

Effects of inoculants Lactobacillus brevis and Lactobacillus parafarraginis on the fermentation characteristics and microbial communities of corn stover silage.

To improve silage quality of crop forages, bacterial inoculants are often employed. In this study, Lactobacillus brevis SDMCC050297 and Lactobacillus parafarraginis SDMCC050300 were used as inoculants to corn stover in lab silos for ensiling. At the initial stage of ensiling, the pH value of the inoculated silages reduced more drastically, and the inoculated silages had higher lactic acid and acetic acid contents. After 20 days of ensiling, a reduction in lactic acid content coupled with an increase in acetic acid and 1,2-propanediol contents was observed in inoculated silages. Furthermore, both the amount of lactic acid bacteria and the abundance of order Lactobacillales in inoculated silages were higher than those of controls in the whole process. Meanwhile, Lb. brevis predominated before day 20 and then the dominance was shifted to Lb. parafarraginis until the late stage of ensiling. In contrast, the epiphytic Lactococcus lactic and Lb. plantarum played major roles at the beginning of naturally fermented silages and then Lb. plantarum and Lb. brevis were the most abundant at the later stage. In conclusion, these two selected strains had capability of improving the silage quality and providing the reproducible ensiling process, thus having the potential as silage inoculants.

Oxygen-Inducible Conversion of Lactate to Acetate in Heterofermentative Lactobacillus brevis ATCC 367.

Lactobacillus brevis is an obligatory heterofermentative lactic acid bacterium that produces high levels of acetate, which improve the aerobic stability of silages against deterioration caused by yeasts and molds. However, the mechanism involved in acetate accumulation has yet to be elucidated. Here, experimental evidence indicated that aerobiosis resulted in the conversion of lactate to acetate after glucose exhaustion in L. brevis ATCC 367 (GenBank accession number NC_008497). To elucidate the conversion pathway, in silico analysis showed that lactate was first converted to pyruvate by the reverse catalytic reaction of lactate dehydrogenase (LDH); subsequently, pyruvate conversion to acetate might be mediated by pyruvate dehydrogenase (PDH) or pyruvate oxidase (POX). Transcriptional analysis indicated that the pdh and pox genes of L. brevis ATCC 367 were upregulated 37.92- and 18.32-fold, respectively, by oxygen and glucose exhaustion, corresponding to 5.32- and 2.35-fold increases in the respective enzyme activities. Compared with the wild-type strain, the transcription and enzymatic activity of PDH remained stable in the Δpox mutant, while those of POX increased significantly in the Δpdh mutant. More lactate but less acetate was produced in the Δpdh mutant than in the wild-type and Δpox mutant strains, and more H2O2 (a product of the POX pathway) was produced in the Δpdh mutant. We speculated that the high levels of aerobic acetate accumulation in L. brevis ATCC 367 originated mainly from the reuse of lactate to produce pyruvate, which was further converted to acetate by the predominant and secondary functions of PDH and POX, respectively.IMPORTANCE PDH and POX are two possible key enzymes involved in aerobic acetate accumulation in lactic acid bacteria (LAB). It is currently thought that POX plays the major role in aerobic growth in homofermentative LAB and some heterofermentative LAB, while the impact of PDH remains unclear. In this study, we reported that both PDH and POX worked in the aerobic conversion of lactate to acetate in L. brevis ATCC 367, in dominant and secondary roles, respectively. Our findings will further develop the theory of aerobic metabolism by LAB.

Characterization of Feruloyl Esterases Produced by the Four Lactobacillus Species: L. amylovorus, L. acidophilus, L. farciminis and L. fermentum, Isolated from Ensiled Corn Stover.

Lactic acid bacteria (LAB) play important roles in silage fermentation, which depends on the production of sufficient organic acids to inhibit the growth of undesirable microorganisms. However, LAB are not able to degrade cellulose and hemicellulose. Bacteria and fibrolytic enzymes are usually used as inoculants to improve the silage quality and digestibility. In the present study, we isolated four Lactobacillus strains (L. amylovorus CGMCC 11056, L. acidophilus CCTCC AB2010208, L. farciminis CCTCC AB2016237 and L. fermentum CCTCC AB2010204) with feruloyl esterase (FAE) activities from ensiled corn stover (CS) by a plate screening assay. The genes encoding FAEs were cloned and hetero-expressed in Escherichia coli. The optimal temperature and pH of these purified enzymes ranged from 45 to 50°C and from 7.0 to 8.0, respectively. They could hydrolyze hydroxycinnamoyl esters in a substrate-specific manner when methyl ferulate, methyl caffeate, methyl ρ-coumarate and methyl sinapinate were used as substrates. Moreover, these four FAEs were able to hydrolyze CS to release hydroxycinnamic acids. Furthermore, these strains could degrade hydroxycinnamic esters, and L. amylovorus CGMCC 11056 was the most efficient strain among these four isolates. These results provided a new target for the development of inoculants to improve silage quality and digestibility.