Inoculation of Yarrowia lipolytica promotes the growth of lactic acid bacteria, Debaryomyces udenii and the formation of ethyl esters in sour meat.

Yarrowia lipolytica N12 and A13 with high lipase activity obtained by mutagenesis were inoculated into sour meat, and their effects on physicochemical properties, microbial community succession, free amino acids, and volatile compounds of sour meat were investigated. Inoculation fermentation increased the contents of free amino acids observably, rapidly reduced pH, promoted the accumulation of total acids, decreased 2-thiobarbituric acid reactive substances (TBARS) values. In addition, the addition of Y. lipolytica might contribute to the growth of lactic acid bacteria, Candida spp., and Debaryomyces udenii, which play an important role in production of volatile compounds. It was shown that inoculation promoted the production of esters, aldehydes, and alcohols, especially ethyl esters, giving sour meat a better meat flavor. Besides, it was found that Y. lipolytica A13 had better fermenting property. Sample of A13 group had higher contents of ethyl esters, free amino acids and dominant microorganisms. The results may help to provide new strains for sour meat fermentation.

Ergothioneine: new functional factor in fermented foods.

Ergothioneine (EGT) is a high-value natural sulfur-containing amino acid and has been shown to possess extremely potent antioxidant and cytoprotective activities. At present, EGT has been widely used in food, functional food, cosmetics, medicine, and other industries, but its low yield is still an urgent problem to overcome. This review briefly introduced the biological activities and functions of EGT, and expounded its specific applications in food, functional food, cosmetic, and medical industries, introduced and compared the main production methods of EGT and respective biosynthetic pathways in different microorganisms. Furthermore, the use of genetic and metabolic engineering methods to improve EGT production was discussed. In addition, the incorporation of some food-derived EGT-producing strains into fermentation process will allow the EGT to act as a new functional factor in the fermented foods.

Combined effects of lipase and Lactiplantibacillus plantarum 1-24-LJ on physicochemical property, microbial succession and volatile compounds formation in fermented fish product.

BACKGROUND: Studies have shown that either the addition of starter culture or enzyme can improve fermentation in fish or other products. However, little research has been carried out on the effects of coupling starter cultures with lipase on the microbial community and product quality. Suanzhayu is a Chinese fermented fish product that mainly relies on spontaneous fermentation, resulting in an unstable flavor and quality. The present study investigated the impact of lipase and Lactiplantibacillus plantarum 1-24-LJ on the quality of Suanzhayu. RESULTS: Inoculation decreased pH and 2-thiobarbituric acid reactive substances (TBARS) values, and also helped the dominance of the strain in the ecosystem, whereas lipase addition raised TBARS values and had little effect on pH, water activity (aw ) and microbiota. The addition of lipase and/or Lpb. plantarum increased the content of alcohols, aldehydes, ketones, esters and umami amino acids. The co-additions with the most significant effect and the total contents of volatile compounds (VCs) and free amino acids (FAAs) were 1801.92 g per 100 g and 21 357.05 mg per 100 g, respectively. Former-Lactobacillus was negatively correlated with pH, aw and Prevotella, but positively with VCs (ethyl ester of heptanoic acid, ethyl ester of octanoic acid) and FAAs (Tyr, Phe). Furthermore, adding Lpb. plantarum 1-24-LJ alone or in combination with lipase shortened the fermentation process. CONCLUSION: The present study provides a recommended Suanzhayu process approach for improving product quality and flavor, as well as shortening fermentation time, by adding Lpb. plantarum 1-24-LJ with or without lipase. © 2023 Society of Chemical Industry.

[A new approach for optimizing empirical prescriptions of famous physicians based on network target: taking Qingluo Decoction as an example].

Based on the network target approach and technology, this study proposed for the first time a novel optimization method for Chinese medicine formulae. Moreover, with Qingluo Decoction as an example, a method for the research and development of Chinese medicine, which combines scientific methodology and experience of famous doctors, was developed. Specifically, based on the composition of Qingluo Decoction, this study used the using network target for intelligent and quantitative analysis on drug actions(UNIQ) to predict the medicinals that targeted the key pathways of rheumatoid arthritis(RA) such as angiogenesis. Then, combining the experience of the first national Chinese medical master LI Ji-ren and Aihui famous Chinese medicine doctor LI Yan and Chinese medicine theory, this study developed a novel angiogenesis-targeted prescription modified Qingluo Decoction(MQLD). Afterward, the clinical efficacy and mechanism of MQLD were verified. The results showed that 27 medicinals with significant regulatory effect on angiogenesis-related key signaling pathways were identified by UNIQ, among which 6 were selected by the Chinese medicine physicians to develop the MQLD. Clinical trials demonstrated that the clinical efficacy of MQLD, in terms of either American College of Rheumatology 20% improvement and 50% improvement criteria(ACR20, ACR50) or TCM syndrome evaluation, was better than that of Qingluo Decoction. Experimental study revealed that MQLD can inhibit RA angiogenesis by acting on the vascular endothelial growth factor(VEGF) pathway, nuclear factor κB(NF-κB) pathway, inflammatory cytokine release, and immune cell regulation. Taken together, this study developed a new formula MQLD with improved clinical efficacy, precise applicable clinical settings, and authorized patent through the network target technology, thus providing a new way for the precise development of Chinese medicine and preservation of the experience of famous physicians.

FordNet: Recommending traditional Chinese medicine formula via deep neural network integrating phenotype and molecule.

Traditional Chinese medicine (TCM) formula is widely used for thousands of years in clinical practice. With the development of artificial intelligence, deep learning models may help doctors prescribe reasonable formulas. Meanwhile, current studies of formula recommendation only focus on the observable clinical symptoms and lack of molecular information. Here, inspired by the theory of TCM network pharmacology, we propose an intelligent formula recommendation system based on deep learning (FordNet), fusing the information of phenotype and molecule. We collected more than 20,000 electronic health records from TCM Master Li Jiren's experience from 2013 to March 2020. In the FordNet system, the feature of diagnosis description is extracted by convolution neural network and the feature of TCM formula is extracted by network embedding, which fusing the molecular information. A hierarchical sampling strategy for data augmentation is designed to effectively learn training samples. Based on the expanded samples, a deep neural network based quantitative optimization model is developed for TCM formula recommendation. FordNet performs significantly better than baseline methods (hit ratio of top 10 improved by 46.9% compared with the best baseline random forest method). Moreover, the molecular information helps FordNet improve 17.3% hit ratio compared with the model using only macro information. Clinical evaluation shows that FordNet can well learn the effective experience of TCM Master and obtain excellent recommendation results. Our study, for the first time, proposes an intelligent recommendation system for TCM formula integrating phenotype and molecule information, which has potential to improve clinical diagnosis and treatment, and promote the shift of TCM research pattern from "experience based, macro" to "data based, macro-micro combined" as well as the development of TCM network pharmacology.

Analysis of carotenoid profile changes and carotenogenic genes transcript levels in Rhodosporidium toruloides mutants from an optimized Agrobacterium tumefaciens-mediated transformation method.

Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium-mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T-DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 105 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and ß-carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1-13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ-carotene and ß-carotene, and the proportion of γ-carotene and ß-carotene were all over 92%. TAIL-PCR was carried out to found T-DNA insertion in these mutants, and insertion hotspot was found. RT-qPCR results showed that CTA1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.

Effects of salt concentration on the quality of paocai, a fermented vegetable product from China.

BACKGROUND: Paocai is a traditional Chinese fermented vegetable food. As the most important ingredient, salt has crucial effects on the bacterial community and volatile compounds of paocai. To demonstrate the effects of salt on the fermentation of paocai, the bacterial composition and volatile compounds were investigated using high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS). RESULTS: The salt had no significant effects on the bacterial community at the phylum level. Proteobacteria and Bacteroidetes gradually decreased during the fermentation, and Firmicutes gradually increased as the dominant bacteria in the late stage of fermentation. At the genus level, Lactobacillus and Lactococcus gradually increased in relative abundance during the fermentation and became the dominant bacteria in paocai. High salt levels can contribute to the growth of Lactobacillus, which became the dominant genus in paocai. The salt concentration affected the profiles of volatile compounds in paocai after fermentation. A total of 42 volatile components were detected by GC-MS, among which phenols, aldehydes, and nitriles were the main ones. A high salt concentration will increase the volatile compound content, mainly aldehydes and alcohols, and improve the flavor of paocai. At the same time, the electronic tongue analysis also showed that a high salt concentration made a major contribution to the flavor of paocai. CONCLUSIONS: These data are helpful to elucidate the effects of salt on the quality of paocai and contribute to improving the quality and reducing the use of salt. © 2021 Society of Chemical Industry.

Effects of salt concentration on microbial diversity and volatile compounds during suancai fermentation.

Suancai is a popular fermented product of Brassica vegetable in China. As important additive, salt concentration has crucial effects on the quality of suancai. To investigate the effects of salt concentration on suancai fermentation, the microbial diversity and volatile compounds (VCs) during fermentation were investigated by using Illumina HiSeq sequencing and GC-MS. Firmicutes, Proteobacteria and Ascomycota were detected as the main phylum during the fermentation with different salt concentrations. Lactobacillus, Lactococcus, Klebsiella, Weissella, Pediococcus, Candida, Cladosporium, Gibberella, Aspergillus, etc., were detected were observed during the fermentation with different concentrations. After fermentation, Lactobacillus predominated the fermentation of suancai and was not affected by salt concentration. Pediococcus, Leuconostoc, Weissella, Sporobolomyces, Azospirillum, Klebsiella, Acinetobacter and Cladosporium were significant affected by salt concentration. Salt addition could affect the VCs profiles and reduce the isothiocyanates after fermentation. Seventy-nine VCs were detected and strongly correlated with the dominant genus Lactobacillus during suancai fermentation. The inoculated fermentation of Lactobacillus could improve the VCs during fermentation. In conclusion, 6% salt addition could acquire a higher Lactobacillus abundance and a better taste quality. These results may facilitate the understanding of the effect of salt concentration on the fermentation ecology to improve suancai characteristics.

Omics-prediction of bioactive peptides from the edible cyanobacterium Arthrospira platensis proteome.

BACKGROUND: Bioinformatics approaches are widely used to evaluate the prospects of novel protein sources in bioactive peptide research. Edible cyanobacteria are considered as potential protein precursors. However, the abundance of unicellular cyanobacterial proteins is largely unknown and highly dynamic according to the cultivation conditions, which need to be considered in this research field. The objective of this work was to evaluate the protein abundance of Arthrospira platensis, as well as to map the bioactive peptide sequences from the high-abundance proteins of the A. platensis proteome. RESULTS: The high-abundance proteins of the A. platensis proteome were identified with a high-performance liquid chromatography-tandem mass spectrometry-based method. A total of 593 proteins were detected and quantified. The occurrence frequency of the bioactive peptides in A. platensis proteome was calculated according to the amino acid sequences via the bioinformatics approaches. Further in silico digested by trypsin, pepsin and chymotrypsin, these proteins liberated 78, 99, and 96 bioactive peptides, respectively. In each case, angiotensin-converting enzyme inhibitors and dipeptidyl peptidase IV inhibitors were enriched. CONCLUSION: This work will help rationally design the protocols for cyanobacterial cultivation, protein pre-treatment and peptide separation, and further produce more peptides with specific functions. © 2017 Society of Chemical Industry.

Characterization the carotenoid productions and profiles of three Rhodosporidium toruloides mutants from Agrobacterium tumefaciens-mediated transformation.

The red yeast Rhodosporidium toruloides is a known lipid producer capable of accumulating large amounts of triacylglycerols and carotenoids. However, it remains challenging to study its carotenoid production profiles owing to limited biochemical information and inefficient genetic tools. Here we used an Agrobacterium tumefaciens-mediated transformation (ATMT) to change its carotenoid production and profiles. We constructed R. toruloides NP11 mutant libraries with ATMT, selected three mutants with different colours, characterized their carotenoid products by high-pressure liquid chromatography-mass spectrometry (HPLC-MS) analysis and assured differences among those strains in terms of carotenoid production and its composition profiles. We then located T-DNA insertion sites using the genome walking technology and provided discussions in terms of the new phenotypes. This study is the first of its kind to change the carotenoid production profiles in R. toruloides. Copyright © 2017 John Wiley & Sons, Ltd.

Protein-protein interaction network of the marine microalga Tetraselmis subcordiformis: prediction and application for starch metabolism analysis.

Under stressful conditions, the non-model marine microalga Tetraselmis subcordiformis can accumulate a substantial amount of starch, making it a potential feedstock for the production of fuel ethanol. Investigating the interactions of the enzymes and the regulatory factors involved in starch metabolism will provide potential genetic manipulation targets for optimising the starch productivity of T. subcordiformis. For this reason, the proteome of T. subcordiformis was utilised to predict the first protein-protein interaction (PPI) network for this marine alga based on orthologous interactions, mainly from the general PPI repositories. Different methods were introduced to evaluate the credibility of the predicted interactome, including the confidence value of each PPI pair and Pfam-based and subcellular location-based enrichment analysis. Functional subnetworks analysis suggested that the two enzymes involved in starch metabolism, starch phosphorylase and trehalose-phosphate synthase may be the potential ideal genetic engineering targets.

Cost-effective production of ergothioneine using Rhodotorula mucilaginosa DL-X01 from molasses and fish bone meal enzymatic hydrolysate.

Ergothioneine (EGT) is a high-value natural antioxidant that cannot be synthesized by the human body. This study showed that Rhodotorula mucilaginosa DL-X01 can use untreated molasses and fish bone meal enzymatic hydrolysate as the substrates to synthesize EGT. By optimizing the growth conditions, the EGT yield reached 29.39 mg/L when molasses and fish bone meal (FBM) were added at 60 g/L and 400 g/L respectively. Finally, the EGT yield was increased to 216.25 mg/L by fed-batch fermentation in a 5 L bioreactor. Compared with the fermentation by yeast extract peptone dextrose medium, the feedstock cost of EGT production was reduced by 330.91 % by using molasses and FBM as substrates. These results showed that R. mucilaginosa DL-X01 can produce high-value EGT using two cheap processing by-products, molasses and FBM, which is of great significance for environmental protection and sustainable development.

Molecular mechanism of saturated aldehyde oxidation: A DFT insight into volatiles forming from decanal thermal oxidation.

Decanal is one of the main products of lipid oxidation. It has been shown that decanal can oxidize to form volatiles with shorter carbon chains during heating, but the mechanism is still unclear. In this study, volatile compounds formed in the decanal thermal oxidation were verified using thermal-desorption cryo-trapping combined with GC-MS. A total of 32 volatile compounds were identified. The oxidation mechanism of decanal was studied by applying density functional theory. Results revealed that the carbonyl carbon atom was the thermal oxidation site of decanal and two pathways of peroxide oxidation were determined: the ortho­carbon and the meta­carbon oxidation. The ortho­carbon oxidation pathway is superior to the occurrence of the meta­carbon oxidation pathway. The oxidative mechanism of decanal was finally summarized as the peroxide oxidation based on radical attack on the carbonyl carbon, which would provide a theoretical basis for exploring the oxidation mechanism of other saturated aldehydes.

Analysis of Fungal Diversity, Physicochemical Properties and Volatile Organic Compounds of Strong-Flavor Daqu from Seven Different Areas.

Strong-flavor Daqu, as a fermentation agent, plays a significant role in shaping the quality of strong-flavor baijius, and fungal species in Daqu are important factors affecting the quality of Daqu. Therefore, we selected strong-flavor Daqu from seven different origins to study the fungal composition and the effects of the fungal composition on the physicochemical properties and volatile organic compounds (VOCs). It was found that the fungal composition influences the physicochemical properties of Daqu. Specifically, there was a positive link between Rhizomucor, Rhizopus, Thermomyces, and liquefying activity and a positive correlation between Aspergillus and fermenting activity. Furthermore, the relationships between esterifying activity and Thermomyces, Rhizomucor, Aspergillus, Pichia, and Saccharomycopsis were found to be positive. The VOCs in Daqu were affected by Aspergillus, Issatchenkia, Pichia, and Thermoascus. Issatchenkia was significantly positively correlated with benzeneethanol as well as Aspergillus and pentadecanoic acid ethyl ester, ethyl myristate. Pichia and Thermoascus were significantly negatively correlated with benzaldehyde and 2-furaldehyde. This study deepens our understanding of the relationship between VOCs, the physicochemical properties with microbial communities, and reference significance for the production of better-quality strong-flavor Daqu.

The effects of Lactiplantibacillus plantarum 3-19 and Pediococcus pentosaceus 18-1 on preventing the accumulation of biogenic amines and promoting the production of volatile organic compounds during sour meat fermentation.

Lactic acid bacteria (LAB) are frequently used in meat fermentation, and mixed stater cultures are reported to perform better than single ones. Lactiplantibacillus plantarum 3-19 and Pediococcus pentosaceus 18-1 were chosen from 28 sour-meat-origin strains to examine the effects of single and combined inoculation on sour meat quality. Natural fermentation was used as a control to investigate changes in pH, water activity (aw), amino acid nitrogen (AN), texture, microbial diversity, and volatile organic compounds (VOCs) during fermentation. The pH and aw of each inoculation group were significantly decreased, and AN content was significantly increased. The inoculation of P. pentosaceus 18-1 significantly reduced putrescine, cadaverine, and tryptamine content (p < 0.05), while the inoculation of Lpb. plantarum 3-19 significantly reduced cadaverine amounts (p < 0.05). At the fermentation endpoint, the total biogenic amines content in the C group was 992.96 ± 14.07, which was 1.65, 2.57, and 3.07 times higher than that in the Lp, Pe, and M groups, respectively. The mixed inoculation group combined the advantages of both strains and decreased total biogenic amines most significantly. At the end of fermentation, the VOCs in C, Lp, Pe, and M groups were 10.11, 11.56, 12.45, and 13.39 times higher than those at the beginning of fermentation. Inoculation promoted the production of key VOCs (OAV > 2000) such as heptanal, octanal, and (E)-2-nonanal. The mixed inoculation group had the highest variety and content of VOCs and the highest content of the above key VOCs, significantly enhancing its fruity, floral, ester, and other aromas. Sensory evaluation indicated that the M group had the best overall acceptability. Finally, it was suggested that a combination of Lpb. plantarum 3-19 and P. pentosaceus 18-1 is a novel and efficient starter culture for processing sour meat since they lower the amounts of biogenic amines in the meat and promote the production of VOCs.

Effects of quercetin- and Lactiplantibacillus plantarum-containing bioactive films on physicochemical properties and microbial safety of grass carp.

In this study, the electrospinning technique was used to co-encapsulate Quercetin (Qu) and Lactiplantibacillus plantarum 1-24-LJ in PVA-based nanofibers, and the effect of bioactive films on fish preservation was evaluated at the first time. The findings indicated that both Lpb. plantarum 1-24-LJ and Qu were successfully in the fibers, and co-loaded fibers considerably outperformed single-loaded fiber in terms of bacterial survival and antioxidant activity. Following fish preservation using the loaded fibers, significant reductions were observed in TVB-N, TBARS, and microbial complexity compared to the control group. Additionally, the co-loaded fibers more effectively reduced the counts of H2S-producing bacteria and Pseudomonas. In the future, fibers with both active substances and LAB hold promise as a novel approach for fish preservation.

Mechanism of thermal oxidation into volatile compounds from (E)-4-decenal: A density functional theory study.

Unsaturated aliphatic aldehyde oxidation plays a significant role in the deep oxidation of fatty acids to produce volatile chemicals. Exposing the oxidation process of unsaturated aliphatic aldehydes is crucial to completely comprehend how food flavor forms. In this study, thermal desorption cryo-trapping in conjunction with gas chromatography-mass spectrometry was used to examine the volatile profile of (E)-4-decenal during heating, and 32 volatile compounds in all were detected and identified. Meanwhile, density functional theory (DFT) calculations were used, and 43 reactions were obtained in the 24 pathways, which were summarized into the peroxide reaction mechanism (ROOH), the peroxyl radical reaction mechanism (ROO·) and the alkoxy radical reaction mechanism (RO·). Moreover, the priority of these three oxidative mechanisms was the RO· mechanism > ROOH mechanism > ROO· mechanism. Furthermore, the DFT results and experimental results agreed well, and the oxidative mechanism of (E)-4-decenal was finally illuminated.

Lactiplantibacillus plantarum A72, a Strain with Antioxidant Properties, Obtained through ARTP Mutagenesis, Affects Caenorhabditis elegans Anti-Aging.

This research endeavored to elucidate the antioxidant attributes of lactic acid bacteria, specifically their impact on anti-aging and lifespan augmentation in Caenorhabditis elegans. The study focused on Lactiplantibacillus plantarum A72, identified through ARTP mutagenesis for its potent antioxidant properties. In vitro analysis affirmed its free radical neutralizing capacity. In C. elegans, the strain not only extended the lifespan by 25.13% and amplified motility 2.52-fold, but also maintained reproductive capabilities. Remarkably, Lpb. plantarum A72 diminished reactive oxygen species (ROS) and malondialdehyde (MDA) levels in C. elegans by 34.86% and 69.52%, respectively, while concurrently enhancing its antioxidant enzyme activities. The strain also bolstered C. elegans survival rates by 46.33% and 57.78% under high temperature and H2O2 conditions, respectively. Transcriptomic scrutiny revealed that Lpb. plantarum A72 could retard C. elegans aging and extend lifespan by upregulating the sod-5 and hsp-16.1 genes and downregulating the fat-6 and lips-17 genes. These findings propose Lpb. plantarum A72 as a potential antioxidant and anti-aging lactic acid bacteria.

Novel microbial fermentation for the preparation of iron-chelating scallop skirts peptides-its profile, identification, and possible binding mode.

Iron chelating peptides have been widely utilized as iron supplements due to their excellent absorption capacity, However, the high cost and cumbersome manufacturing process of these peptides significantly limit their industrial application. In this study, fermentation was used for the first time to prepare iron chelating peptides. Bacillus altitudinis 3*1-3 was selected as the most suitable strain from 50 strains. The hydrolysates of fermented scallop skirts showed excellent iron-chelating capacity (9.39 mg/g). Aspartic acid, glutamic acid, and histidine are crucial for the binding of peptides to ferrous ions. The heptapeptide (FEDPEFE) forms six binding bonds with ferrous irons. Compared with ferrous sulfate, peptide-ferrous chelate showed more stability in salt solution and simulated gastrointestinal juice (p < 0.05). Furthermore, the fermentation method could save >50% of the cost compared with the enzymatic method. The results can provide a theoretical basis for the preparation of ferrous-chelated peptides using the fermentation method.

Inhibition Mechanism of Lactiplantibacillus plantarum on the Growth and Biogenic Amine Production in Morganella morganii.

Morganella morganii, a spoilage bacterium in fermented foods, produces harmful biogenic amines (BAs). Although Lactiplantibacillus plantarum is widely used to inhibit spoilage bacteria, the inhibition pattern and inhibition mechanism of M. morganii by Lpb. plantarum are not well studied. In this study, we analysed the effects of the addition of Lpb. plantarum cell-free supernatant (CFS) on the growth and BA accumulation of M. morganii and revealed the mechanisms of changes in different BAs by using RNA sequencing transcriptome analysis. The results showed that Lpb. plantarum CFS could significantly inhibit M. morganii BAs in a weak acid environment (pH 6), and the main changes were related to metabolism. Carbohydrate and energy metabolism were significantly down-regulated, indicating that Lpb. plantarum CFS inhibited the growth activity and decreased the BA content of M. morganii. In addition, the change in histamine content is also related to the metabolism of its precursor amino acids, the change in putrescine content may also be related to the decrease in precursor amino acid synthesis and amino acid transporter, and the decrease in cadaverine content may also be related to the decrease in the cadaverine transporter. The results of this study help to inhibit the accumulation of harmful metabolites in fermented foods.