Identification of microorganisms in wet coffee fermentation Coffea arabica Var Catimor and Castillo in Jardín, Antioquia-Colombia, using culture-dependent methods / Identificación de microorganismos en fermentación húmeda de café Coffea arabica Var Catimor y Castillo en Jardín, Antioquia-Colombia, usando métodos cultivo-dependientes

Background: Mild Colombian coffees are recognized worldwide for their high-quality coffee cup. However, there have been some failures in post-harvest practices, such as coffee grain fermentation. These failures could occasionally lead to defects and inconsistencies in quality products and economic losses for coffee farmers. In Colombia, one of the fermentation methods most used by coffee growers is wet fermentation, conducted by submerging the de-pulped coffee beans for enough time in water tanks to remove the mucilage. Objectives: We evaluated the effect of the water (g)/de-pulped coffee (g) ratio (I: 0/25, II: 10/25, III: 20/25) and final fermentation time (24, 48, and 72 hours) on the total number of microbial groups. We also identified microorganisms of interest as starter cultures. Methods: We used a completely randomized experimental design with two factors; the effect of the water (g)/de-pulped coffee (g) ratio (I: 0/25, II: 10/25, III: 20/25) and final fermentation time (24, 48, and 72 hours), for 9 treatments with two replicates. During the coffee fermentation (1,950 g), the pH and °Brix were monitored. Total counts of different microbial groups (mesophiles, coliforms, lactic-acid bacteria, acetic-acid bacteria, and yeasts) were performed. Various isolates of microorganisms of interest as starter cultures (lactic-acid bacteria and yeasts) were identified using molecular sequencing techniques. Results: 21 lactic-acid bacteria (LAB) isolates and 22 yeasts were obtained from the different mini-batch fermentation systems. The most abundant lactic-acid bacteria species found were Lactiplantibacillus plantarum (46%) and Levilactobacillus brevis (31%). Pichia kluivery (39%) and Torulaspora delbrueckii (22%) were the most abundant yeast species. Conclusion The studied factors did not have effect over the microorganism's development. The identified bacterial and yeasts species have potential as starter cultures for better-quality coffees and in fermentation-related applications.

Antecedentes: Los cafés suaves lavados colombianos son reconocidos a nivel mundial por su buena puntuación sensorial; sin embargo, se han detectado fallas en las prácticas de postcosecha, como lo es la fermentación de los granos de café. Dichas fallas pueden causar defectos y carecer de consistencia en la calidad del producto, ocasionando pérdidas económicas para los caficultores. En Colombia, uno de los métodos más usados por los caficultores es la fermentación húmeda, la cual consiste en sumergir los granos de café despulpado en tanques con agua por un período de tiempo que permita la remoción del mucílago. Objetivos: La presente investigación evaluó la incidencia que tienen la proporción agua/granos despulpados de café (I: 0/25, II: 10/25, III: 20/25) y el tiempo final de fermentación (24, 48 y 72 horas) en el recuento final de grupos microbianos. Por otra parte, se identificaron taxonómicamente microorganismos de interés para su uso como cultivos iniciadores. Métodos: Mini-lotes consistieron en café despulpado (1950 g) puesto en recipientes de plástico abiertos y sumergidos en agua. Se aplicó un diseño experimental completamente aleatorizado de dos factores (proporción agua/ granos de café despulpado y tiempo) a tres niveles, para un total de nueve tratamientos con dos replicas. Durante las fermentaciones de café (1,950 g), el pH y los grados ºBrix, fueron monitoreados. Se realizaron los recuentos totales de los diferentes grupos microbianos: mesófilos, coliformes, bacterias ácido-lácticas, bacterias ácido-acéticas y levaduras. Se identificaron molecularmente diferentes aislados con potencial para ser usados como cultivos iniciadores (bacterias ácido-lácticas y levaduras). Resultados: Los resultados obtenidos mostraron que no hubo diferencia estádisticamente significativa entre los tratamientos aplicados y el recuento final de microorganismos. Un total de 21 aislados de bacterias ácido-lácticas (BAL) y 22 levaduras lograron obtenerse a partir de los diferentes sistemas de fermentación en mini-lote. Las especies de bacterias ácido-lácticas con mayor porcentaje acorde a su identificación taxonómica, corresponden a Lactiplantibacillus plantarum (46%), Levilactobacillus brevis (31%). Las especies de levaduras con mayor porcentaje acorde a su identificación taxonómica corresponden a Pichia kluivery (39%) y Torulaspora delbrueckii (22%). Conclusión Los factores estudiados no afectaron el crecimiento de ninguno de los grupos microbianos presentes en la fermentacion del café. Las especies de microorganismos identificados tienen potencial para se usados como cultivos starter o en aplicaciones dentro de las ciencias de fermentación.

Age Discrimination Based on Volatile Components of Arisaema Cum Bile / 中国实验方剂学杂志

ObjectiveTo discriminate the age of Arisaema Cum Bile， the combination of headspace solid-phase microextraction （HS-SPME） with gas chromatography-mass spectrometry （GC-MS） was applied to explore the differences of volatile components of unfermented， 1-year fermented， 2-year fermented， and 3-year fermented Arisaema Cum Bile. MethodSamples with different fermentation durations were collected and HS-SPME-GC-MS technology was employed to detect the volatile components of each sample. The relative contents of detected volatile components were processed and analyzed by chemometrics methods such as principal component analysis （PCA）， hierarchical cluster analysis （HCA）， and partial least squares discriminant analysis （PLS-DA）. ResultThe results showed that 145 volatile components were identified. Among these volatile components， the relative contents of heterocyclic， alcohols， aldehydes and aromatics were high. PCA， HCA， and PLS-DA can effectively separate Arisaema Cum Bile with four different ages. Based on variable importance in projection （VIP） value > 1， 73 markers of differential volatile components were identified. The content of 2，6，11-trimethyldodecane and m-xylene in unfermented samples was the highest， and the content difference between them and those in fermented samples was significant （P<0.05）. 2，3-butanediol was detected only in 1-year samples， octane was detected only in 2-year samples， and ethyl heptanoate was detected only in 3-year samples. These components can be used as odor markers for Arisaema Cum Bile with different fermentation years. ConclusionThe identification method of volatile components of Arisaema Cum Bile was established by HS-SPME-GC-MS technology， which can realize the rapid identification of unfermented， 1-year fermented， 2-year fermented， and 3-year fermented samples， and provide a scientific basis for the standardization of processing technology and quality standards of Arisaema Cum Bile.

Identificación y selección de Saccharomyces cerevisiae nativas para mejorar el proceso productivo del Pisco a partir de uva Quebranta

El Pisco es el destilado del Perú, elaborado a partir de mostos recientemente fermentados con uvas criollas denominadas "pisqueras". Las levaduras son los microorganismos clave en la fermentación y el uso de cepas nativas seleccionadas presenta ventajas competitivas para la tipicidad del producto, así como también para la estandarización del producto y el control microbiológico del proceso. El objetivo fue identificar y seleccionar las cepas de levaduras nativas para la producción del Pisco de uva Quebranta aisladas de procesos productivos de Pisco en el valle de Ica. Para ello, se emplearon técnicas microbiológicas y moleculares mediante el análisis de ITS1-5.8S-ITS2 PCR-RFLP para la identidad taxonómica. La evaluación de las cepas para producir Pisco consistió en el análisis fisicoquímico y organoléptico del destilado obtenido con las cepas seleccionadas. Se evaluaron 3 aislados para la producción de Pisco identificados como Saccharomyces cerevisiae: UNA SC - 25, UNA SC - 49 y UNA SC - 54, de los cuales la cepa UNA SC-49 destacó por mostrar aptitudes enológicas diferentes a las otras cepas. Este trabajo constituye el primer registro de levaduras nativas del Perú para mostos procedentes de uva Quebranta.

El Pisco stands as Peru's distilled spirit, crafted from recently fermented musts derived from native grape varieties known as "pisqueras". Yeasts serve as decisive microorganisms in the fermentation process, and the utilization of selected native strains confers competitive advantages for product typicity, standardization, and microbiological control within the production process. The aim was to identify and select native yeast strains for Quebranta grape Pisco production, isolated from Pisco production processes in the Ica Valley. Microbiological and molecular techniques were employed, utilizing ITS1-5.8S-ITS2 PCR-RFLP analysis for taxonomic identification. The assessment of yeast strains for Pisco production involved the physicochemical and organoleptic analysis of the distilled product obtained using the selected strains. Three isolates were evaluated for Pisco production, identified as Saccharomyces cerevisiae: UNA SC - 25, UNA SC - 49, and UNA SC - 54. Among these, the UNA SC-49 strain stood out due to displaying oenological characteristics distinct from the other strains. This work is the first documentation of native yeasts in Peru for musts derived from Quebranta grapes.

Reduction of Microbial Contamination of Chechin nchabe (Cooked Fermented Cassava) Produced in Noun (West Cameroon) by a Second Fermentation

The Chechim nchabe is a traditional food widely consumed in Foumban, Foumbot, Koutaba, Massangam, Kouoptamo, Malentouen and Magba, 07 Departments of Noun (West region of Cameroon). It is obtained by fermenting cassava sticks cooked on the surface of river or spring water. Unfortunately, the bad hygienic quality of the environment during production promotes its contamination by pathogenic germs. The objective of this study is to carry out a second fermentation in order to reduce contamination of Chechim nchabe by pathogens germs during production. To achieve this objective, a survey on the socio-economic data, profile of the producers, production protocol and characteristics of product have been realized. After microbiological analysis of Chechim nchabe, a second fermentation was performed in the laboratory. From the results, it appears that all the producers are women, aged between 51 and 58 years and 87% of them not attending school. The water used for soaking the cassava revealed that 54% of women use river water and 46% spring water. The Chechim nchabe samples collected after traditional production in the 07 Departments of Noun, show average contamination of Enterobacteriaceae, moulds, staphylococci, Escherichia coli and lactic acid bacteria with respective concentrations of 4.7; 4.1; 4.4; 4.7 and 4.8 Log10ufc/mL. However, Chechim nchabe produced in urban areas such as Foumbot and Foumban recorded low contamination compared to that produced in rural areas like Massangam, which were heavily contaminated with Escherichia coli and Enterobacteriaceae. It was also noted that the Chechim nchabe produced in spring water is more contaminated than that produced in river water. The second fermentation for 10 hours of Chechim nchabe in a basin, after 12 hours of traditional fermentation, eliminated all of pathogenic germs from Chechim nchabe. This second fermentation of 10 hours could be a solution to guarantee the sanitary quality of Chechim nchabe before its consumption.

Análises bromatológicas de missô artesanal / Bromatological analysis of handmade misso

Objetivo ­ Realizar análises bromatológicas de umidade, proteínas, pH e cinzas em missôs artesanais. A fermentação é um processo biotecnológico que tem sido utilizado para modificar e produzir alimentos desde a antiguidade. Nas últimas duas décadas, o interesse nos efeitos benéficos dos fermentados na saúde humana aumentou e tornou essa categoria de alimentos cada vez mais popular principalmente no Oriente. No mercado há uma ampla variedade de pastas à base de soja fermentada por microorganismos sendo conhecido popularmente como missô. Métodos ­ As análises realizadas foram secagem direta em estufa a 105°C graus para determinação da umidade (%) e calcinação em mufla para cinzas (%), determinação de pH por meio do peagâmetro e análise de proteínas através do teste de Biureto. Resultados ­ No presente estudo as amostras obtiveram um teor de umidade entre 52,71% a 60,48%, teor de cinzas variando de 1,12% a 22,7%, pH entre 5,35 e 8,68, e um teor de proteínas variando de 11,1% a 13,2%. Discussão ­ Foi interpretado e comparado os resultados obtidos com as análises de outros estudos, além disso, apontado algumas questões do campo bromatológico das pesquisas dos estudos comparados e as limitações do presente trabalho. Conclusão ­ O processo fermentativo de alimentos com microorganismos resulta em um produto diferenciado que pode ser benéfico a saúde com diferentes características organolépticas. Nossos resultados foram parcialmente semelhantes com outras pesquisas sendo que

Fermentative losses and lactic acid content of elephant grass silages added with macaúba cake

Elephant grass is indicated for silage production but requires additives to increase dry matter content because it reduces the production of effluents, potentially improves the fermentation pattern, and preserves the nutrients of the silage. This study aimed to evaluate the effects of including macaúba cake in elephant grass ensilage on dry matter content, lactic acid bacteria population, lactic acid production, pH values, losses by gases and effluents, and dry matter recovery. The experimental design was completely randomized, in a 3x6 factorial scheme, with three levels of inclusion of macaúba cake (0, 10, and 20%) and six opening times (1, 5, 10, 20, 40, and 60 days after ensilage), with four repetitions. Macaúba cake was an effective moisture-absorbing additive, increasing dry matter content, lactic acid bacteria population, and lactic acid content and reducing the pH. The losses by effluents and gases decreased, and dry matter recovery increased with the addition of this biodiesel co-product. The 20% level of inclusion of macaúba cake in elephant grass ensilage allowed for better preserving the ensiled material.

Screening of lysine production by Escherichia coli and Klebsiella isolates at different carbon sources

Lysine is an essential amino acid that is not biologically manufactured in the body. Different chemical methods for lysine production are expensive and give low yields. The present study was conducted with the purpose to evaluate the biochemical production of lysine by different carbon sources using bacterial isolates. Three carbon sources namely glucose, sucrose, and fructose were used to evaluate the biochemical production of lysine by Escherichia coli and Klebsiella spp. isolates. Optimum incubation periods were between 48-96 hours. An extensive amount of lysine was produced by all of these isolates in L6 fermentation medium. Maximum lysine was produced by Klebsiella isolate K1 6.48 g/L after 96 hours of incubation by using glucose as carbon source followed by 6.0 g/L by Klebsiella isolates K3 after 72 hours of incubation when sucrose was used as a carbon source at 37 °C. Highest amount of lysine was produced at 96 hours by Klebsiella isolates in addition to E. coli. From all three carbon sources using Klebsiella isolates and E. coli, glucose showed better lysine production.

Multicellular coupling fermentation for 3'-sialyllactose conversion using N-acetyl-glucosamine and lactose / 生物工程学报

Sialyllactose is one of the most abundant sialylated oligosaccharides in human milk oligosaccharides (HMOs), which plays an important role in the healthy development of infants and young children. However, its efficient and cheap production technology is still lacking presently. This study developed a two-step process employing multiple-strains for the production of sialyllactose. In the first step, two engineered strains, E. coli JM109(DE3)/ pET28a-BT0453 and JM109(DE3)/pET28a-nanA, were constructed to synthesize the intermediate N-acetylneuraminic acid. When the ratio of the biomass of the two engineered strains was 1:1 and the reaction time was 32 hours, the maximum yield of N-acetylneuraminic acid was 20.4 g/L. In the second step, E. coli JM109(DE3)/ pET28a-neuA, JM109(DE3)/ pET28a-nst and Baker's yeast were added to the above fermentation broth to synthesize 3'-sialyllactose (3'-SL). Using optimal conditions including 200 mmol/L N-acetyl-glucosamine and lactose, 150 g/L Baker's yeast, 20 mmol/L Mg2+, the maximum yield of 3'-SL in the fermentation broth reached 55.04 g/L after 24 hours of fermentation and the conversion rate of the substrate N-acetyl-glucosamine was 43.47%. This research provides an alternative technical route for economical production of 3'-SL.

Secretory expression and fermentation optimization for extracellular production of pullulanase in Vibrio natriegens / 生物工程学报

Pullulanase is a starch debranching enzyme, which is difficult in secretory expression due to its large molecular weight. Vibrio natriegens is a novel expression host with excellent efficiency in protein synthesis. In this study, we achieved secretory expression of the full-length pullulanase PulA and its truncated mutant PulN2 using V. natriegens VnDX strain. Subsequently, we investigated the effects of signal peptide, fermentation temperature, inducer concentration, glycine concentration and fermentation time on the secretory expression. Moreover, the extracellular enzyme activities of the two pullulanases produced in V. natriegens VnDX and E. coli BL21(DE3) were compared. The highest extracellular enzyme activity of PulA and PulN2 in V. natriegens VnDX were 61.6 U/mL and 64.3 U/mL, which were 110% and 62% that of those in E. coli BL21(DE3), respectively. The results indicated that V. natriegens VnDX can be used for secretory expression of the full-length PulA with large molecular weight, which may provide a reference for the secretory expression of other large molecular weight proteins in V. natriegens VnDX.

Efficient synthesis of L-methionine by engineering the one carbon module of Escherichia coli / 生物工程学报

L-methionine, also known as L-aminomethane, is one of the eight essential amino acids required by the human body and has important applications in the fields of feed, medicine, and food. In this study, an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain (Escherichia coli W3110 ΔIJAHFEBC/PAM) previously constructed in our laboratory. Firstly, the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase, MetF) and screening of hydroxymethyltransferase (GlyA) from different sources, optimizing the one-carbon module. Subsequently, cysteamine lyase (hydroxymethyltransferase, MalY) and cysteine internal transporter gene (fliY) were overexpressed to improve the supply of L-homocysteine and L-cysteine, two precursors of the one-carbon module. The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L, and up to 18.26 g/L in a 5 L fermenter. The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine, and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module. This study may facilitate further improvement of microbial fermentation production of L-methionine.

Bioconversion of C1 gases and genetic engineering modification of gas-utilizing microorganisms / 生物工程学报

C1 gases including CO, CO2 and CH4, are mainly derived from terrestrial biological activities, industrial waste gas and gasification syngas. Particularly, CO2 and CH4 are two of the most important greenhouse gases contributing to climate change. Bioconversion of C1 gases is not only a promising solution to addressing the problem of waste gases emission, but also a novel route to produce fuels or chemicals. In the past few years, C1-gas-utilizing microorganisms have drawn much attention and a variety of gene-editing technologies have been applied to improve their product yields or to expand product portfolios. This article reviewed the biological characteristics, aerobic or anaerobic metabolic pathways as well as the metabolic products of methanotrophs, autotrophic acetogens, and carboxydotrophic bacteria. In addition, gene-editing technologies (e.g. gene interruption technology using homologous recombination, group Ⅱ intron ClosTron technology, CRISPR/Cas gene editing and phage recombinase-mediated efficient integration of large DNA fragments) and their application in these C1-gas-utilizing microorganisms were also summarized.

Optimization of the medium and fermentation condition for the Penicillium aurantiocandidum Z12 strain with molluscicidal actions against Oncomelania hupensis / 中国血吸虫病防治杂志

Objective To optimize the culture and fermentation conditions of the Penicillium aurantiocandidum Z12 strain, a fungal strain with molluscicidal actions against Oncomelania hupensis, so as to provide the basis for the research and development of molluscicidal active substances from the P. aurantiocandidum Z12 strain and its fermentation broth and large-scale fermentation. Methods The carbon source, nitrogen source and mineral salts were identified in the optimal culture medium for the P. aurantiocandidum Z12 strain with a single-factor experiment to determine the best fermentation condition for the P. aurantiocandidum Z12 strain. Factors that significantly affected the growth of the P. aurantiocandidum Z12 strain were identified using the Plackett-Burman design, and the best range of each factor was determined using the steepest climb test. Response surface analyses of temperature, pH value, seeding amount and liquid-filling quantity were performed using the Box-Behnken design to create a regression model for fermentation of the P. aurantiocandidum Z12 strain to identify the optimal culture medium. Results Single-factor experiment preliminarily identified the best culture medium and conditions for the P. aurantiocandidum Z12 strain as follows: sucrose as the carbon source at approximately 20 g/L, tryptone as the nitrogen source at approximately 5 g/L, K2HPO4 as the mineral salt at approximately 5 g/L, initial pH at approximately 8, temperature at approximately 28 °C, seeding amount at approximately 6%, and liquid-filling quantity at approximately 50 mL/100 mL. Plackett-Burman design showed that factors that significantly affected the growth of the P. aurantiocandidum Z12 strain included temperature (t = −5.28, P < 0.05), seeding amount (t = 5.22, P < 0.05), pH (t = −4.30, P < 0.05) and liquid-filling quantity (t = −4.39, P < 0.05). Steepest climb test showed the highest mycelial growth at pH of 7.5, seeding amount of 8%, and liquid-filling quantity of 40 mL/100 mL, and this condition was selected as the central point of response surface analysis for the subsequent optimization of fermentation conditions. Response surface analyses using the Box-Behnken design showed that the optimal conditions for fermentation of the P. aurantiocandidum Z12 strain included sucrose at 15 g/L, tryptone at 5 g/L, K2HPO4 at 5 g/L, temperature at 28.2 °C, pH at 7.5, seeding amount at 10%, and liquid-filling quantity at 35.8 mL/100.0 mL, resulting in 0.132 g yield of the P. aurantiocandidum Z12 strain. Conclusion The optimal culture condition for the P. aurantiocandidum Z12 strain has been identified, and the optimized culture medium and fermentation condition may effectively improve the fermentation yield of the P. aurantiocandidum Z12 strain.

Varieties systematization and standards status analysis of fermented Chinese medicine / 中国中药杂志

Fermented Chinese medicine has long been used. Amid the advance for preservation of experience, the connotation of fermented Chinese medicine has been enriched and improved. However, fermented Chinese medicine prescriptions generally contain a lot of medicinals. The fermentation process is complicated and the conventional fermentation conditions fail to be strictly controlled. In addition, the judgment of the fermentation end point is highly subjective. As a result, quality of fermented Chinese medicine is of great difference among regions and unstable. At the moment, the quality standards of fermented Chinese medicine are generally outdated and different among regions, with simple quality control methods and lacking objective safe fermentation-specific evaluation indictors. It is difficult to comprehensively evaluate and control the quality of fermented medicine. These problems have aroused concern in the industry and also affected the clinical application of fermented Chinese medicine. This article summarized and analyzed the application, quality standards, and the modernization of fermentation technology and quality control methods of fermented Chinese medicine and proposed suggestions for improving the quality standards of the medicine, with a view to improving the overall quality of it.

Difference of lipid-lowering efficacy of "Xinjianqu" before and after fermentation and its mechanism based on LKB1-AMPK pathway and 16S rDNA sequencing technology / 中国中药杂志

On the basis of establishing the prescription of Xinjianqu and clarifying the increase of the lipid-lowering active ingredients of Xinjianqu by fermentation, this paper further compared the differences in the lipid-lowering effects of Xinjianqu before and after fermentation, and studied the mechanism of Xinjianqu in the treatment of hyperlipidemia. Seventy SD rats were randomly divided into seven groups, including normal group, model group, positive drug simvastatin group(0.02 g·kg~(-1)), and low-dose and high-dose Xinjianqu groups before and after fermentation(1.6 g·kg~(-1) and 8 g·kg~(-1)), with ten rats in each group. Rats in each group were given high-fat diet continuously for six weeks to establish the model of hyperlipidemia(HLP). After successful modeling, the rats were given high-fat diet and gavaged by the corresponding drugs for six weeks, once a day, to compare the effects of Xinjianqu on the body mass, liver coefficient, and small intestine propulsion rate of rats with HLP before and after fermentation. The effects of Xinjianqu before and after fermentation on total cholesterol(TC), triacylglyceride(TG), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol(LDL-C), alanine aminotransferase(ALT), aspartate aminotransferase(AST), blood urea nitrogen(BUN), creatinine(Cr), motilin(MTL), gastrin(GAS), and the Na~+-K~+-ATPase levels were determined by enzyme-linked immunosorbent assay(ELISA). The effects of Xinjianqu on liver morphology of rats with HLP were investigated by hematoxylin-eosin(HE) staining and oil red O fat staining. The effects of Xinjianqu on the protein expression of adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK), phosphorylated AMPK(p-AMPK), liver kinase B1(LKB1), and 3-hydroxy-3-methylglutarate monoacyl coenzyme A reductase(HMGCR) in liver tissues were investigated by immunohistochemistry. The effects of Xinjianqu on the regulation of intestinal flora structure of rats with HLP were studied based on 16S rDNA high-throughput sequencing technology. The results showed that compared with those in the normal group, rats in the model group had significantly higher body mass and liver coefficient(P<0.01), significantly lower small intestine propulsion rate(P<0.01), significantly higher serum levels of TC, TG, LDL-C, ALT, AST, BUN, Cr, and AQP2(P<0.01), and significantly lower serum levels of HDL-C, MTL, GAS, Na~+-K~+-ATP levels(P<0.01). The protein expression of AMPK, p-AMPK, and LKB1 in the livers of rats in the model group was significantly decreased(P<0.01), and that of HMGCR was significantly increased(P<0.01). In addition, the observed＿otus, Shannon, and Chao1 indices were significantly decreased(P<0.05 or P<0.01) in rat fecal flora in the model group. Besides, in the model group, the relative abundance of Firmicutes was reduced, while that of Verrucomicrobia and Proteobacteria was increased, and the relative abundance of beneficial genera such as Ligilactobacillus and Lachnospiraceae＿NK4A136＿group was reduced. Compared with the model group, all Xinjianqu groups regulated the body mass, liver coefficient, and small intestine index of rats with HLP(P<0.05 or P<0.01), reduced the serum levels of TC, TG, LDL-C, ALT, AST, BUN, Cr, and AQP2, increased the serum levels of HDL-C, MTL, GAS, and Na~+-K~+-ATP, improved the liver morphology, and increased the protein expression gray value of AMPK, p-AMPK, and LKB1 in the liver of rats with HLP and decreased that of LKB1. Xinjianqu groups could regulate the intestinal flora structure of rats with HLP, increased observed＿otus, Shannon, Chao1 indices, and increased the relative abundance of Firmicutes, Ligilactobacillus(genus), Lachnospiraceae＿NK4A136＿group(genus). Besides, the high-dose Xinjianqu-fermented group had significant effects on body mass, liver coefficient, small intestine propulsion rate, and serum index levels of rats with HLP(P<0.01), and the effects were better than those of Xinjianqu groups before fermentation. The above results show that Xinjianqu can improve the blood lipid level, liver and kidney function, and gastrointestinal motility of rats with HLP, and the improvement effect of Xinjianqu on hyperlipidemia is significantly enhanced by fermentation. The mechanism may be related to AMPK, p-AMPK, LKB1, and HMGCR protein in the LKB1-AMPK pathway and the regulation of intestinal flora structure.

Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae / 中国中药杂志

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.

Genetic modification of acetogens and optimization of fermentation process in C1-gas bioconversion / 生物工程学报

The current linear economy model relies on fossil energy and increases CO2 emissions, which contributes to global warming and environmental pollution. Therefore, there is an urgent need to develop and deploy technologies for carbon capture and utilization to establish a circular economy. The use of acetogens for C1-gas (CO and CO2) conversion is a promising technology due to high metabolic flexibility, product selectivity, and diversity of the products including chemicals and fuels. This review focuses on the physiological and metabolic mechanisms, genetic and metabolic engineering modifications, fermentation process optimization, and carbon atom economy in the process of C1-gas conversion by acetogens, with the aim to facilitate the industrial scale-up and carbon negative production through acetogen gas fermentation.

Metabolic engineering of Escherichia coli for adipic acid production / 生物工程学报

Adipic acid is a high-value-added dicarboxylic acid which is primarily used in the production of nylon-66 for manufacturing polyurethane foam and polyester resins. At present, the biosynthesis of adipic acid is hampered by its low production efficiency. By introducing the key enzymes of adipic acid reverse degradation pathway into a succinic acid overproducing strain Escherichia coli FMME N-2, an engineered E. coli JL00 capable of producing 0.34 g/L adipic acid was constructed. Subsequently, the expression level of the rate-limiting enzyme was optimized and the adipic acid titer in shake-flask fermentation increased to 0.87 g/L. Moreover, the supply of precursors was balanced by a combinatorial strategy consisting of deletion of sucD, over-expression of acs, and mutation of lpd, and the adipic acid titer of the resulting E. coli JL12 increased to 1.51 g/L. Finally, the fermentation process was optimized in a 5 L fermenter. After 72 h fed-batch fermentation, adipic acid titer reached 22.3 g/L with a yield of 0.25 g/g and a productivity of 0.31 g/(L·h). This work may serve as a technical reference for the biosynthesis of various dicarboxylic acids.

Variations of glucose content in Massa Medicata Fermentata during processing based on quantitative proton nuclear magnetic resonance / 中国中药杂志

A quantitative proton nuclear magnetic resonance(qHNMR) method was established to determine the glucose content in commercially available Massa Medicata Fermentata(MMF) products and explore the variations of glucose content in MMF products during processing. The qHNMR spectrum of MMF in deuterium oxide was obtained with 2,2,3,3-d_4-3-(trimethylsilyl) propionate sodium salt as the internal standard substance. With the doublet peaks of terminal hydrogen of glucose with chemical shift at δ 4.65 and δ 5.24 as quantitative peaks, the content of glucose in MMF samples was determined. The glucose content showed a good linear relationship within the range of 0.10-6.44 mg·mL~(-1). The relative standard deviations(RSDs) of precision, stability, repeatability, and recovery for determination were all less than 2.3%. The glucose content varied in different commercially available MMF samples, which were associated with the different fermentation days, wheat bran-to-flour ratios, and processing methods. The glucose content in MMF first increased and then decreased over the fermentation time. Compared with the MMF products fermented with wheat bran or flour alone, the products fermented with both wheat bran and flour had increased glucose. The glucose content of bran-fried MMF was slightly lower than that of raw MMF, while the glucose content in charred MMF was extremely low. In conclusion, the qHNMR method established in this study is simple, fast, and accurate, serving as a new method for determining the glucose content in MMF. Furthermore, this study clarifies the variations of glucose content in MMF during processing, which can not only indicate the processing degree but also provide a scientific basis for revealing the fermentation mechanism and improving the quality control of MMF.

Screening,identification,enzymatic characteristics and optimization of fermentation condition of a high alkaline xylanase-producing strain / 中国生物制品学杂志

@#ObjectiveTo screen a high alkaline xylanase-producing strain,subject to molecular identification and characterization of enzymatic property,and optimize its fermentation condition.MethodsThe farmland soil samples from Shanxi,Henan and Zhejiang Provinces were collected aseptically,from which the high xylanase-producing strains were screened by enrichment culture,isolation and identification of 16S r DNA,and determined for enzymatic properties.The carbon source(xylan,lactose,soluble starch,sucrose,bran and glucose),nitrogen source[ammonium oxalate,peptone,yeast powder,(NH_4)_2SO_4,NH_4Cl,Na NO_3,urea,beef extract,bean powder,KNO_3,(NH_4)_2HPO_4or random mixture of two of peptone,yeast powder,beef extract,(NH_4)_2HPO_4and bean powder],metal ion[CuCl_2,MgCl_2,ZnCl_2,Al_2(SO_4)_3,CoCl_2,MnCl_2,AgNO_3,NaCl,CaCl_2,FeCl_2,BaCl_2 and FeCl_3],pH value(3.0～10.0)in medium and the fermentation temperature(25,28,30,33,35,37 and 40℃)were optimized by single factor test,while the contents of components[xylan,(NH_4)_2HPO_4and bean powder]by response surface method.The high alkaline xylanase-producing strain was fermented by using the optimized medium under the optimized condition,and determined for xylanase activity,and the result was compared with that predicated in model.ResultsA high alkaline xylanase-producing strain named as SX6-18 was screened and identified as Paemibacillus based on 16s rDNA sequence analysis.The xylanase produced by the strain maintained more than 70%of relative activity at temperatures of 25～55℃and pH 6.0～10.0.Mg(2+)promoted while Fe(2+)promoted while Fe(3+),Mn(3+),Mn(2+)and Cu(2+)and Cu(2+)inhibited the activity of xylanase.The optimal carbon source,nitrogen source and metal ion of the medium were xylan,bean powder+(NH_4)_2HPO_4 and Fe(2+)inhibited the activity of xylanase.The optimal carbon source,nitrogen source and metal ion of the medium were xylan,bean powder+(NH_4)_2HPO_4 and Fe(3+),while the optimal p H value and temperature for fermentation were 8.0 and 30℃，respectively.However,the optimal component contents were 15.00 g/L xylan,3.03 g/L(NH_4)_2HPO_4and 3.28 g/L bean powder.The activity of xylanase cultured in optimized medium under opti-mized condition for fermentation reached 701.08 U/m L,which was closed to the expected value(693.96 U/m L).ConclusionA high alkaline xylanase-producing strain SX6-18 was successfully screened,which maintained relatively high enzyme activity in alkaline condition.This study laid a foundation of application of xylanase in papermaking and washing fields.

Modulating the production of xylanase by Bacillus pumilus BS131 through optimization using waste fiber sludge / Modulando a produção de xilanase por Bacillus pumilus BS131 por meio da otimização usando lodo de fibra residual

In recent days, cheapest alternative carbon source for fermentation purpose is desirable to minimize production cost. Xylanases have become attractive enzymes as their potential in bio-bleaching of pulp and paper industry. The objective of the present study was to identify the potential ability on the xylanase production by locally isolated Bacillus pumilus BS131 by using waste fiber sludge and wheat bran media under submerged fermentation. Culture growth conditions were optimized to obtain significant amount of xylanase. Maximum xylanase production was recorded after 72 hours of incubation at 30 °C and 7 pH with 4.0% substrate concentration. In the nutshell, the production of xylanase using inexpensive waste fiber sludge and wheat-bran as an alternative in place of expensive xylan substrate was more cost effective and environment friendly.

Nos últimos dias, a fonte alternativa de carbono mais barata para fins de fermentação é desejável para minimizar o custo de produção. As xilanases têm se tornado enzimas atraentes como seu potencial no biobranqueamento da indústria de papel e celulose. O objetivo do presente estudo foi identificar a capacidade potencial na produção de xilanase por Bacillus pumilus BS131 isolado localmente usando lodo de fibra residual e farelo de trigo em meio de fermentação submersa. As condições de crescimento da cultura foram otimizadas para obter uma quantidade significativa de xilanase. A produção máxima de xilanase foi registrada após 72 horas de incubação a 30 °C e pH 7 com concentração de substrato de 4,0%. Resumindo, a produção de xilanase usando lodo de fibra residual de baixo custo e farelo de trigo como uma alternativa no lugar do substrato de xilano caro foi mais econômica e ecológica.