Effect of proteolytic bacteria on texture and colour quality of khanom-jeen, traditional Thai fermented rice noodles.

Khanom-jeen are traditional Thai fermented rice noodles with a characteristic flavour and texture. Based on their ability to hydrolyse rice proteins, five strains of proteolytic bacteria were isolated from fermented rice flour used to produce noodles in Thailand. Enterobacter ludwigii strain SK01 showed the highest degree of rice protein hydrolysis among the five strains and was selected as a starter culture for khanom-jeen production. The viable count of micro-organisms, acidity, pH, and protein and starch contents were compared throughout the fermented rice production with and without the SK01 strain. The pH of fermented rice increased on the first day of solid-state fermentation and subsequently decreased to 3·7-3·8 under both conditions. The protein content of rice significantly decreased from 80 to 18·2 ± 0·5 mg g-1 and 26 ± 0·6 mg g-1 during fermentation with and without the SK01 strain, respectively. The tensile strength, breaking length, brightness, and whiteness, which are important factors for the texture and appearance of khanom-jeen, were improved using the SK01 strain to ferment rice. The results of this laboratory-scale study indicated that the use of the proteolytic SK01 strain in fermented rice production can improve the quality of khanom-jeen noodles. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of Enterobacter ludwigii strain SK01 as a proteolytic bacterial starter culture improved the quality of khanom-jeen noodles. Compared with khanom-jeen noodles produced without E. ludwigii strain SK01, those produced using this strain showed increased tensile strength, breaking length, brightness and whiteness. These findings will be useful for the development of specific and controllable methods for producing khanom-jeen noodles with desirable texture and colour.

Proteinivorax hydrogeniformans sp. nov., an anaerobic, haloalkaliphilic bacterium fermenting proteinaceous compounds with high hydrogen production.

A search for the organisms responsible for the degradation of biomass of primary producers in Tanatar lakes resulted in the isolation of a novel anaerobic, haloalkaliphilic microorganism, strain Z-710T. The strain grows on proteinaceous substrates (peptides) but not on proteins. A rather limited range of substances of other classes can be utilised together with tryptone but not individually. An interesting physiological feature of the novel strain is a high capacity for hydrogen production (up to 30% v/v) during proteolytic fermentation. Phylogenetic analysis based on the 16S rRNA gene sequence similarity revealed that the organism can be assigned to the previously described genus Proteinivorax. According to its physiological features and the low DNA-DNA hybridisation level of the strain with the type strain of the only previously described Proteinivorax species-Proteinivorax tanatarense Z-910T-strain Z-710T is described here as representing a novel species with the name Proteinivorax hydrogeniformans sp. nov. The type strain is Z-710T (= DSM 102085T = VKM B-3042T).

Alkaline and Halophilic Protease Production by Bacillus luteus H11 and Its Potential Industrial Applications.

This paper presents the results of the study on the production of protease by Bacillus luteus H11 isolated from an alkaline soda lime. B. luteus H11 was identified as an alkalohalophilic bacterium, and its extracellular serine endoprotease also showed an extreme alkali- and halotolerance. It was remarkably stable in the presence of NaCl up to 5 M. The enzyme was active in a broad range of pH values and temperatures, with an optimum pH of 10.5 and a temperature of 45 °C. It had a molecular mass of about 37 kDa and showed activity against azocasein and a synthetic substrate for the subtilisin-like protease, N-succinyl-l-phenylalanine-p-nitroanilide. The halo-alkaline protease produced by B. luteus H11 seems to be significant from an industrial perspective because of its tolerance towards high salinity and alkalinity as well as its stability against some organic solvents, surfactants and oxidants. These properties make the protease suitable for applications in food, detergent and pharmaceutical industries, and also in environmental bioremediation.

Anoxynatronum buryatiense sp. nov., an anaerobic alkaliphilic bacterium from a low mineralization soda lake in Buryatia, Russia.

An anaerobic alkaliphilic, proteolytic bacterium, strain Su22T, was isolated from the bottom sediment of the alkaline low mineralization lake Sulphatnoe (Selenginsky district, Buryatia, Russia). A comparative analysis of the 16S rRNA gene sequence revealed that this bacterium was closely related to Anoxynatronum sibiricum Z-7981T with a similarity of 98.1 %. Strain Su22T differed from A. sibiricum Z-7981T in its inability to use carbohydrates, peptone and amino acids as carbon sources. Strain Su22T grew over a temperature range of 20-40 °C with an optimum at 30 °C and within the pH range 7.4-11.0 with an optimum at pH 9.6. Sodium cations stimulated the growth of the strain considerably with an optimal concentration at 0.76-1.09 M. The whole-cell fatty acid profile included C16 : 1ω7c, C16 : 0 and C16 : 0 ALDE. The G+C content was 46.1 mol%. Based on the DNA-DNA hybridization level (53.2 %) and phenotypical differences between strains Su22T and Z-7981T, the new isolate is thus considered to represent a novel species, for which the name Anoxynatronumburyatiense sp. nov. is proposed. The type strain is Su22Т (=VKM B-2510T=CECT 8731T).

Microbiological quality of raw milk attributable to prolonged refrigeration conditions.

Refrigerated storage of raw milk is a prerequisite in dairy industry. However, temperature abused conditions in the farming and processing environments can significantly affect the microbiological quality of raw milk. Thus, the present study investigated the effect of different refrigeration conditions such as 2, 4, 6, 8, 10 and 12 °C on microbiological quality of raw milk from three different dairy farms with significantly different initial microbial counts. The bacterial counts (BC), protease activity (PA), proteolysis (PL) and microbial diversity in raw milk were determined during storage. The effect of combined heating (75 ± 0·5 °C for 15 s) and refrigeration on controlling those contaminating microorganisms was also investigated. Results of the present study indicated that all of the samples showed increasing BC, PA and PL as a function of temperature, time and initial BC with a significant increase in those criteria ≥6 °C. Similar trends in BC, PA and PL were observed during the extended storage of raw milk at 4 °C. Both PA and PL showed strong correlation with the psychrotrophic proteolytic count (PPrBC: at ≥4 °C) and thermoduric psychrotrophic count (TDPC: at ≥8 °C) compared to total plate count (TPC) and psychrotrophic bacterial count (PBC), that are often used as the industry standard. Significant increases in PA and PL were observed when PPrBC and TDPC reached 5 × 104 cfu/ml and 1 × 104 cfu/ml, and were defined as storage life for quality (S LQ), and storage life for safety (S LS) aspects, respectively. The storage conditions also significantly affected the microbial diversity, where Pseudomonas fluorescens and Bacillus cereus were found to be the most predominant isolates. However, deep cooling (2 °C) and combination of heating and refrigeration (≤4 °C) significantly extended the S LQ and S Ls of raw milk.

Sustainable leather tanning: Enhanced properties and pollution reduction through crude protease enzyme treatment.

In this study, proteolytic bacteria, particularly Pseudomonas aeruginosa strain SM4 (OQ349573), were isolated from tannery solid waste dumping yard soil and employed to produce extracellular protease enzymes. The bacteria exhibited optimal growth after 30 h of incubation at 37 °C and pH 7. Under conditions of 55 °C, pH 8, and a substrate concentration of 2 %, the crude enzyme displayed its highest activity at 105 UmL-1. Notably, the produced crude enzyme showed no discernible inhibitory effects on detergents, metal salts, or organic solvents. Application of the crude protease at concentrations of 3 % and 2 % in chrome tanning of goatskins (GS) and cowhides (CH), respectively, yielded significant reductions of 35 % and 30 % in chromium and other post-tanning chemicals compared to conventional processes. Despite the 30 to 35 % reduction in tanning and post-tanning chemicals, the uptake of chrome and associated chemicals by crust leather was higher than observed in conventional processes. Chromium content analysis of the effluent revealed an 81 % reduction during piloting in real industrial operations, accompanied by reductions of about 46 % in BOD and COD pollution loads. The finished leather obtained from the enzymatic process exhibited superior mechanical properties, including higher tensile strengths (210 and 195 kg cm-2), stitch tear (92 and 165 kg cm-1), grain crack load (28 and 32 kg), and distension (73 and 62 mm) for GS and CH, respectively, surpassing or closely aligning with standard values and those obtained in conventional processes.

A pilot study of alterations of the gut microbiome in canine chronic kidney disease.

Introduction: Gut dysbiosis has been noted in humans and animals with chronic kidney disease (CKD). However, little is known about the gut microbiome in canine patients with CKD. This study aimed to analyze and compare the gut microbiome profiles of healthy and CKD dogs, including differences in the gut microbiome between each CKD stage. Methods: The study was conducted on 29 client-owned dogs who underwent physical examination, complete blood count (CBC), serum biochemistry, and urinalysis. The gut microbiome profile of healthy dogs (n = 10) and dogs with CKD (n = 19) was analyzed employing 16S rRNA sequencing. Results: Significant differences were seen in the composition of the gut microbiome, with increased operational taxonomic units from the phylum Proteobacteria (p = 0.035), family Enterobacteriaceae (p < 0.001), and genus Enterococcus (p = 0.002) in dogs with CKD, and a decrease in the genus Ruminococcus (p = 0.007). Furthermore, an increase in both the progression of CKD and abundance of genus Klebsiella (Jonckheere-Terpstra test statistic value (JT) = 2.852, p = 0.004) and Clostridium (JT = 2.018, p = 0.044) was observed. Discussion: Our study demonstrated that in dogs with CKD, the composition of the gut microbiome varied depending on the stage of CKD. Alterations in gut microbiome composition observed in CKD patients are characterized by an increase in proteolytic bacteria and a decrease in saccharolytic bacteria. These findings suggest specific gut microbiota could be targeted for clinical management of uremic dogs with CKD.

Improving the nutritional value and bioactivity of soybean meal in solid-state fermentation using Bacillus strains newly isolated from the gut of the termite Termes propinquus.

The present study aimed to isolate and characterize proteolytic Bacillus spp. from termite guts to test the possibility of application for improving the nutritional value and bioactivity of fermented soybean meal (FSBM). Aerobic endospore-forming bacteria were isolated from the gut of the termite Termes propinquus. Ten isolates with high levels of soy milk degradation were selected and tested for extracellular enzyme production. Among them, two isolates, Tp-5 and Tp-7, exhibited all tested hydrolytic enzyme activities (cellulase, xylanase, pectinase, amylase, protease, lipase and phytase), weak alpha hemolytic and also antagonistic activities against fish pathogenic species of Aeromonas and Streptococcus. Both phylogenetic and biochemical analyses indicated that they were closely related to Bacillus amyloliquefaciens. During solid-state fermentation of SBM, Tp-5 and Tp-7 exhibited the highest protease activity (1127.2 and 1552.4 U g-1, respectively) at 36 h, and the resulting FSBMs showed a significant increase in crude protein content and free radical-scavenging ability (P < 0.05), as well as an improvement in the composition of amino acids, metabolites and other nutrients, while indigestible materials such as fiber, lignin and hemicellulose were decreased. The potential strains, especially Tp-7, improved the nutritional value of FSBM by their strong hydrolytic and antioxidant activities, together with reducing antinutritional components.

Microbial decomposition of crustacean shell for production of bioactive metabolites and study of its fertilizing potential.

Crustacean shell waste disposal is considered as biggest problem in seafood processing centers. Incineration and landfilling are the commonest ways of disposal of the waste which causes environmental pollution. Microbial bio-conversion is one of the promising approaches to minimize the wastes by utilizing the same for deriving different value added metabolites. In this perspective, chitinase- and protease-producing bacterial strains were isolated from shrimp culture pond, and the potent isolate was subsequently identified as Alcaligenes faecalis SK10. Fermentative optimization of the production of chitinase (85.42 U/ml), protease (58.57 U/ml), and their catalytic products, viz., N-acetylamino sugar (84 µg/ml) and free amino acids (112 µg/ml), were carried out by utilizing shrimp and crab shell powder as principal substrate. The fermented hydrolysate (FH) was subsequently applied to evaluate its potential to be a candidate fertilizer for the growth of leguminous plant Pisum sativum and Cicer arietinum, and the results were compared with chitin, chitosan, and commercial biofertilizer amended group. The results revealed that FH have paramount potential to improve plants morpho-physiological parameters like stem and root length, chlorophyll, cellular RNA, protein content, and soil physico-chemical parameters like total nitrogen, magnesium, calcium, phosphorus, and potassium significantly (p < 0.05). Moreover, the application of FH also selectively encouraged the growth of free-living nitrogen-fixing bacteria, Rhizobium, phosphate-solubilizing bacteria in the soil by 4.82- and 5.27-, 5.57- and 4.71, and 7.64- and 6.92-fold, respectively, in the rhizosphere of P. sativum and C. arietinum, which collectively is a good sign for an ideal biofertilizer. Co-supplementation of FH with commercial PGPR-biofertilizer significantly influenced the morpho-physiological attributes of plant and physico-chemical and microbial attributes of soil. The study validated proficient and sustainable utilization of fermented hydrolysate of waste crustacean shell as biofertilizer.

Breastmilk Lipids and Oligosaccharides Influence Branched Short-Chain Fatty Acid Concentrations in Infants with Excessive Weight Gain.

SCOPE: The aim is to identify breastmilk components associated with fecal concentration of SCFAs and to investigate whether they differ between infants with high weight gain (HW) and normal weight gain (NW). METHODS AND RESULTS: Breastmilk and fecal samples are collected from mother-infant dyads with HW (n = 11) and NW (n = 15) at 5 and 9 months of age. Breastmilk is profiled on ultra-performance LC-quadrupole TOF-MS platform. Fecal SCFAs are quantified using an isotope-labeled chemical derivatization method. Human milk oligosaccharides (HMOs) are quantified using HPLC after fluorescent derivatization. Lower levels of α-linolenic acid, oleic acid, 3-oxohexadecanoic acid, LPE (P-16:0), LPC (16:0), LPC (18:0), PC (36:2) in breastmilk from mothers from the HW-group at 5 months of age is found. Fecal SCFA concentrations are increased during the transition period from breastfeeding to complementary feeding. Fecal butyrate concentration is higher in the NW-group at 9 months of age. Fecal branched SCFAs are positively associated with breastmilk phospholipid levels, free-fatty acid levels, HMO-diversity, sialylated-HMOs, 6'-sialyllactose, and disialyl-lacto-N-hexaose. CONCLUSION: Fecal branched SCFA concentrations seem to be affected by breastmilk lipid and HMO composition. These differences in breastmilk metabolites may partially explain the excessive weight gain in early life.

In situ identification of the synthrophic protein fermentative Coprothermobacter spp. involved in the thermophilic anaerobic digestion process.

Thermophilic bacteria have recently attracted great attention because of their potential application in improving different biochemical processes such as anaerobic digestion of various substrates, wastewater treatment or hydrogen production. In this study we report on the design of a specific 16S rRNA-targeted oligonucleotide probe for detecting members of Coprothermobacter genus characterized by a strong protease activity to degrade proteins and peptides. The newly designed CTH485 probe and helper probes hCTH429 and hCTH439 were optimized for use in fluorescence in situ hybridization (FISH) on thermophilic anaerobic sludge samples. In situ probing revealed that thermo-adaptive mechanisms shaping the 16S rRNA gene may affect the identification of thermophilic microorganisms. The novel developed FISH probe extends the possibility to study the widespread thermophilic syntrophic interaction of Coprothermobacter spp. with hydrogenotrophic methanogenic archaea, whose establishment is a great benefit for the whole anaerobic system.

Diversity of the cultivable human gut microbiome involved in gluten metabolism: isolation of microorganisms with potential interest for coeliac disease.

Gluten, a common component in the human diet, is capable of triggering coeliac disease pathogenesis in genetically predisposed individuals. Although the function of human digestive proteases in gluten proteins is quite well known, the role of intestinal microbiota in the metabolism of proteins is frequently underestimated. The aim of this study was the isolation and characterisation of the human gut bacteria involved in the metabolism of gluten proteins. Twenty-two human faecal samples were cultured with gluten as the principal nitrogen source, and 144 strains belonging to 35 bacterial species that may be involved in gluten metabolism in the human gut were isolated. Interestingly, 94 strains were able to metabolise gluten, 61 strains showed an extracellular proteolytic activity against gluten proteins, and several strains showed a peptidasic activity towards the 33-mer peptide, an immunogenic peptide in patients with coeliac disease. Most of the strains were classified within the phyla Firmicutes and Actinobacteria, mainly from the genera Lactobacillus, Streptococcus, Staphylococcus, Clostridium and Bifidobacterium. In conclusion, the human intestine exhibits a large variety of bacteria capable of utilising gluten proteins and peptides as nutrients. These bacteria could have an important role in gluten metabolism and could offer promising new treatment modalities for coeliac disease.

Influenza virus and proteolytic bacteria co-infection in respiratory tract from individuals presenting respiratory manifestations / Vírus influenza e bactéria proteolítica co-infectantes em trato respiratório de indivíduos com manifestações respiratórias

A role for proteolytic bacteria in the exacerbation of influenza virus has been shown in natural hosts such as pigs and humans. Four hundred seven samples were collected from the respiratory tract of individuals presenting clinical manifestations, during influenza season (2003-2005) in São Paulo City. The aim of this study was to evaluate the incidence of determined bacteria co-infecting virus in human respiratory tract. Tests, such as bacteriological, immunofluorescence (IF), RT/PCR and hemagglutination (HA) were used for bacterial and viral investigation. Thirty seven (9.09 percent) positive for influenza virus were screened by IF. The RT/PCR confirmed the presence of influenza virus in these samples. Bacterial and agar casein tests demonstrated that 18 (48.64 percent) individuals were infected with proteolytic bacteria such as Staphylococcus spp., Streptococcus spp. and Pseudomonas spp. Among these samples, 13 (35.13 percent) were co-infected with influenza A virus. Influenza type B, co-infecting bacteria were found in five (13.51 percent) samples. In vitro the S. aureus protease increased the influenza HA titer after contact for 30 min at 25 ºC. Results revealed the occurrence of co-infection with proteolytic bacteria and influenza in the evaluated individuals. This finding corroborates that virus versus bacteria synergism could be able to potentiate respiratory infection, increasing damage to hosts.

O papel da bactéria proteolítica na exacerbação do vírus influenza tem sido demonstrado em hospedeiros naturais como porcos e humanos. Foram coletadas 407 amostras do trato respiratório de indivíduos apresentando manifestações clínicas, durante a estação da influenza (2003-2005) na cidade de São Paulo. Este trabalho teve como objetivo avaliar a incidência de determinadas bactérias que junto com vírus co-infectarem o trato respiratório humano. Testes bacteriológicos, e virológicos como imunofluorescência (IF), RT/PCR e hemaglutinação (HA) foram usados nas investigações viral e bacteriana. Pelo teste de IF foram selecionadas trinta e sete (9,09 por cento) amostras positivas para o vírus influenza. A presença do vírus influenza foi confirmada pela técnica de RT/PCR. Pelos testes bacteriológicos e do agar caseina, verificou-se que 18 (48,64 por cento) dos indivíduos foram infectados com bactérias proteolíticas tais como Staphylococcus spp., Streptococcus spp. e Pseudomonas spp. Destas amostras, 13 (35,13 por cento) foram co-infectadas com vírus influenza tipo A, e 5 (13,51 por cento) com influenza tipo B. No experimento in vitro com influenza e S. aureus, detectou-se aumento do título hemaglutinante deste vírus, após contacto de 30 min a 25 ºC. Os resultados obtidos revelaram a ocorrência de co-infecção com bactéria proteolítica e vírus influenza nos indivíduos avaliados. Estes achados corroboram com a investigação do sinergismo, entre bactéria e vírus, que poderia ser capaz de potencializar infecção respiratória, aumentando os riscos aos hospedeiros.