Utilización de desperdicios de camarón para recuperación de quitina, proteínas y pigmentos por vía microbiana

La fermentación láctica ha sido utilizada desde tiempos inmemoriales para aumentar la vida de anaquel de los alimentos, debido principalmente a la producción de ácidos orgánicos y compuestos con actividad antimicrobiana. En la presente tesis se estudió el uso de la fermentación láctica del desecho de camarón para la recuperación de productos de alto valor agregado, tales como quitina, pigmentos y proteasas. En una primera etapa se establecieron las condiciones de la fermentación mediante la selección del iniciador, probando dos cultivos comerciales de Christian Hansen: Floracarn SL (Lactobacillus pentosus y Staphylococcus carnosus) y LP- 1 (Lactobacillzrs pentoszts), así como cepas aisladas de camarones tropicales identificadas como Lactobacillus casei (A3) y Lactobacillus sp. (B2). La cepa B2 y Lactobacillus pentosus produjeron concentraciones más altas de ácido láctico, aunque en las fermentaciones con Lb. pentoms se detectaron bajas concentraciones de ácido acético, de ahí que la cepa B2 fuera elegida para la determinación de las concentraciones de la fbente de carbono (glucosa) y nivel de inoculación. Las concentraciones de glucosa e inóculo con las que se obtuvieron mejores resultados heron 10% (p/p base húmeda) y 5% (v/p base húmeda), respectivamente. Para la elección de las condiciones antes mencionadas e realizaron análisis estadístico de varianza así como superficies de respuesta. El uso de iniciador y la concentración inicial de glucosa heron factores determinantes en la fermentación láctica de desechos de camarón. En este sentido, altas concentraciones de glucosa e iniciador redujeron el tiempo de fermentación, e incrementaron la concentración del ácid0 láctico producido. La inoculación modificó el patrón fermentativo de hetero a homoláctico La microflora mostró una reducción inicial de enterobacterias, sin embargo después de 8 horas la cuenta de coliformes aumentó, mientras que la de bacterias Iácticas fue constante durante todo el tiempo de fermentación y solo hacia el final del cultivo disminuyó. La segunda etapa consistió en el aislamiento de los productos: proteasas, quitina y pigmentos, a partir del ensilado obtenido después de 48 horas. Se separaron dos fracciones una sólida y otra líquida, las cuales heron de un 55 a 60% y de 40 a 45 YO del peso original en base húmeda, respectivamente. El sólido o sedimento consistió principalmente de quitina, proteína, pigmentos y minerales, en un 2 1.23, 21.73, 0.0123 y 8.21% (p/p base seca) respectivamente. La purificación parcial de las proteasas a partir del ensilado fie llevada a cabo mediante ultrafiltración y cromatografia de permeación en gel (GPC). La fracción soluble de la fermentación he filtrada y centrifigada, y el líquido liofilizado para su almacenamiento. Posteriormente el polvo fue resuspendido y ultrafiltrado, su concentración y fraccionamiento fueron hechos probando diferentes membranas de 300, 50, 10 y 2 kDa. Las concentraciones de proteína y actividad proteolítica en caseína pH 7 y hemoglobina pH 2, heron determinadas en el permeado y retenido de cada ultrafiltración realizada, observándose que había una mayor concentración de proteínas con actividad utilizando membranas de 2 y 10 ma, no encontrándose diferencias significativas entre éstas. Sin embargo con la membraria de 2 el proceso era muy lento, por lo que se eligió el retenido obtenido con la de 10 kDa. Las fracciones de la ultrafiltración mostraron actividad en condiciones ácidas y alcalinas. El peso molecular de las fracciones con actividad fie determinado mediante (3% correspondiendo a 13, 26 y 56 kDa; la actividad específica he mayor a pH neutro que a ácido. El retenido que fie la fracción que presentó mayor actividad he aplicada en una etapa subsecuente para la desproteinización de quitina. Por vía fermentativa se removió un 75.7 YO de proteína y 65.80 YO de calcio de la quitina cruda (sedimento). Posteriofmente, se ralizaron tratamientos químicos para llevar a cabo despigmentación, desmineralización y desproteinización, para este último tratamiento se probaron proteasas comerciales y proteasas aisladas del camarón (retenido). Para la desproteinización enzimática se procedió a determinar el tiempo de reacción con tripsina bovina comercial durante 96 horas, encontrándose un aumento en la proteína soluble dukante las primeras 48 horas, y después de ese tiempo la concentración de proteína soluble se mantuvo estable. Por lo que se fijó un tiempo de 48 horas para estudios con las otras enzimas, proteasas de camarón, mezcla de enzimas: Savinasa, Neutrasa, Alcalasa y Esperasa (Novo, Copenhague, Dinamarca). Asimismo para comparar fueron obtenidas quitinas a partir del desecho sin fermentar y se analizaron productos comerciales. Análisis elemental y espectroscopia de infrarrojo por transformada de Fourier heron empleadas para caracterizar las quitinas obtenidas. Los resultados indicaron que los productos de la fermentación Iáctica heron similares a los producidos por métodos tradicionales.Ensilation of prawn waste allows the recovery of added value products such as chitin, pigments and proteins, which could be used for animal feed, and other applications. Ensilation is defined as a conservation process in which acids, added or produced, inhibit the growth of pathogen microorganisms. In a fermented silage acid is produced in sit21 by lactic acid bacteria, using carbohydrate sources. In this study the fermentation conditions and selection of microorganism were determined based on acid production, glucose concentration as carbohydrate source, and inoculum level. Afterwards the fermentation pattern was determined. From the various lactic acid bacteria tested, Lactobacillus yerltoszts and Lactobacillus sp. (B2) were the best acid-producing microorganisms. However small quantities of acetic acid were detected in samples inoculated with Lactobacillus pentosns, therefore B2 was chosen for the determination of glucose and inoculum levels; the best results were obtained at 10% (w/w w.b.1 and 5% (viw w.b.), respectively. The use of starters and the initial glucose concentration were critical factors in prawn head fermentation. High initial glucose and starter concentrations reduced fermentation time and increased the amount of lactic acid produced. Inoculation modified the fermentation from heterofermentative to homofermentative. There was an initial reduction of enterobacteria, but after 8 hours of fermentation coliform counts increased whereas lactic acid bacteria counts were constant during most of the fermentation time, and decreased at the end. The above described conditions produced a stable silage, with two fractions: solid and liquid The next stage was the recovery of products: partial purification of proteases was done from the liquid by means of ultrafiltration and gel permeation chromatography. Molecular weight proteases ranged at 13, 26 and 56 kDa, specific activity was higher at pH 7 than 2 Finally the chitin and pigments were extracted from the solid, 21 and 0.012 YO (wiw wet basis): respectively. Chitin was isolated from the silage solid after demineralisation with hydrochloric acid and deproteination with alkali. Enzymatic deproteination was carried out using commercial proteases (Alcalase, Pronase, Savlnase and Esperase) and proteases from the fermented prawn waste. Chitin FTIR spectra and elemental analysis were comparable with commercial products. K-qwords: prcrwn wastes, lactic acid fermentation, chitin, Lactobacillw, proteases, pigmerlts, protein silage

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Preparation of biological fish silage and its effect on the performance and meat quality characteristics of quails (Coturnix coturnix japonica)

The aim of the present study was to produce fish silage by lactic acid fermentation and evaluate its use in feeding of quails (Coturnix coturnix japonica). An oven-dried mixture of fish silage and soybean meal (1:1 w/w) was used to prepare the diets with different levels of inclusion (0, 10, 20 and 30%) and evaluate its effect on the performance and meat quality of 160 quails. The inclusion level did not affect the growth and feed conversion ratio. The carcass yield (70.3%) and sensory quality of breast meat were not significantly different among the treatments (p&gt;0.05). However, the concentration of unsaturated fatty acids such as oleic (C18:1n9C), linoleic (C18:2n6C), linolenic (C18:3n3), arachidonic (C20:4n6), cis eicosapentaenoic (C20:5n3) and cis docosahexaenoic (C22:6n3) increased in quail breast meat with the inclusion of fish silage:soybean mixture in the diet (p<0.05). Fish silage and its use in quail diets could offer a good alternative for fish waste utilization as feedstuff component for the improvement of fatty acid composition in its breast meat