A STUDY ON SUITABILITY OF FOUR ENRICHMENT BROTHS FOR PCR-BASED DETECTION OF LISTERIA MONOCYTOGENES FROM RAW MEAT

Four enrichment broths were evaluated for their compatibility with the polymerase chain reaction (PCR) for detection of Listeria monocytogenes from raw meat after single‐step enrichment. Standardized PCR protocols for listeriolysin O (hlyA) gene were used for the species‐specific identification of L. monocytogenes. Four broths, namely, modified University of Vermont broth (MUVM), Listeria enrichment broth (LEB), Fraser broth (FB) and polymyxin, acriflavin, lithium chloride, ceftazidime, aesculin, mannitol, egg yolk broth (PALCAM) , were inoculated with L. monocytogenes. The enriched cultures were subjected for PCR. Similarly, meat samples were artificially spiked with various concentrations of L. monocytogenes, these spiked samples were enriched in the above‐mentioned four broths and subjected to PCR to determine the medium that was most compatible for PCR‐based detection of L. monocytogenes. The aliquots taken during different incubation periods were subjected to three different procedures for the concentration of the target organism for use in PCR. Results revealed that MUVM was better than other broths for the detection of L. monocytogenes by both PCR and cultural method; moreover, it was able to support the growth of as low as 10 cfu/g of meat. Concentration of the target organisms by centrifugation and washing with PCR buffer was the most suitable method for improving PCR performance for detection of L. monocytogenes. Goat (n = 67) and buffalo (n = 45) meat samples from local markets were also screened by both PCR and cultural method to validate the results obtained from the spiking studies. Both results were in agreement in spiking studies as well as screening of market meat samples.     

CLONING, EXPRESSION AND CHARACTERIZATION OF NOVEL LECTIN FROM ORYZA SATIVA

A lectin gene homolog of Oryza sativa was successfully cloned and expressed in Escherichia coli. The deduced amino acid sequence of the protein product showed a significant similarity with known chitin‐binding lectins. Most of the recombinant lectin was found in an insoluble aggregated form as inclusion bodies and only a small part was in the culture medium in a soluble active form. Functional recombinant lectin was recovered from the inclusion bodies by solubilization with 8 M urea in Tris/HCl buffer, pH 7.0 and renaturation by 10‐fold dilution in the same buffer. The recombinant lectin with His‐tag was simply purified to homogeneity by the process of affinity chromatography and was obtained with a yield of 6–8 mg/L culture. The recombinant lectin was a homo‐dimer composed of 22 kDa. The hemagglutination activity of the recombinant lectin was optimal at pH 4.0–7.0 and it was very sensitive to inhibition by N‐acetylneuraminic acid and thyroglobulin.     

APPLICATION OF RESPONSE SURFACE METHODOLOGY TO OPTIMIZE DOCOSAHEXAENOIC ACID PRODUCTION BY SCHIZOCHYTRIUM sp. S31

The optimum concentrations of nutrients (glucose and yeast extract) and cultivation conditions (concentration of sodium chloride, pH and incubation time) on docosahexaenoic acid (DHA) production by Schizochytrium sp. S31 in flasks at 30C were studied. Experiment design employed fractional factorial design, path of steepest ascent, central composite design and response surface methodology (RSM). The empirical model developed by RSM was adequate to describe the relationships between the studied factors and the response of DHA production. Based on contour plots and canonical analysis, the optimal conditions for maximizing DHA production (516 mg/L) were at 27.98 g glucose/L, 4.52 g yeast extraction/L, 24.82 g sodium chloride/L, pH 6.96 and incubation for 4 days at 30C. Experimental verification of the optimal conditions resulted in about 97% of the predicted DHA production by the model.     

Evaluation Antibacterial Activity of Quaternary‐Based Chitin/Chitosan Derivatives In Vitro

Abstract: Total of 3 water‐soluble quaternary‐based chitin/chitosan derivatives, which have an identical molecular weight and anion, were synthesized and characterized. Their antibacterial activities against Salmonella cholerae‐suis and Bacillus subtilis were evaluated in vitro. The polysaccharides exhibited the antibacterial efficiency. Their minimum inhibitory concentration (MIC) values vary from 0.02 to 20.48 mg/mL, and their minimum bactericidal concentration (MBC) values vary from 0.08 to 40.96 mg/mL against S. cholerae‐suis and B. subtilis, respectively. Futhermore, the extent of Bacillus subtilis cells damage was examined via transmission electron microscopy (TEM) to show how N,N,N‐trimethylchitosan (TMC) gradually destroyed and killed B. subtilis cells when they were treated with TMC. One of those quaternary polymers, O‐([2‐hydroxy‐3‐trimethylammonium])propyl chitin (OHT‐chitin), which can be directly and easily synthesized from chitin in bulk quantities, also was demonstrated its antibacterial activity. These water soluble quaternary‐based chitin/chitosan derivatives that have antibacterial effect should be potentially used as antimicrobial agents in many fields. Practical Application: The main practical application behind the investigation and evaluation antibacterial activity of 3 water‐soluble quaternary‐based chitin/chitosan derivatives could be potentially used as antimicrobial agents in many fields. These polysaccharides represent a renewable source of natural biodegradable polymers and meet with the emergence of more and more food safe problems.     

OXYRASETM ENZYME AND MOTILITY ENRICHMENT FUNG-YU TUBE FOR RAPID DETECTION OF LISTERIA MONOCYTOGENES AND LISTERIA SPECIES1

A rapid and simple method using a U-shaped glass apparatus (Fung-Yu tube) for early determination of the presence of Listeria monocytogenes and Listeria species in mixed cultures and inoculated meat samples has been developed. This system utilizes unique biochemical and physical properties of Listeria for selective enrichment. Fraser broth was used as a selective enrichment broth especially for observation of esculin hydrolysis (blackening of broth), and semisolid Modified Oxford agar was used for selective detection of motility of Listeria. When Fung-Yu tubes containing 0.1 unit/mL of Oxyrase^TM (membrane fractions of Escherichia coli) were inoculated with L. monocytogenes, an enhanced early growth of L. monocytogenes occurred. A presumptive positive result for low numbers of L. monocytogenes (1–100 CFU/g) in the presence of large numbers of competitive microflora in pre-enriched (24 h) ground beef samples using the Fung-Yu tube method with the aid of Oxyrase^TMwas obtainable within 10 h. Using this system, isolation of Listeria in the presence of mixed bacterial flora (44 species), such as Bacillus, Escherichia, Klebsiella, Proteus, Salmonella, Shigella, Staphylococcus, and Streptococcus, and in inoculated ground beef was successful in 24–48 h. The Fung-Yu tube procedure is a highly sensitive, selective, and easy-to-use method to separate and isolate L. monocytogenes and other Listeria spp. from other contaminating microorganisms in meats.     

Preparation and characteristics of squid pen β‐chitin prepared under optimal deproteinisation and demineralisation condition

Squid (Todarodes pacifica) pen was an excellent source of β‐chitin with 25.5% yield. The optimal condition to prepare squid pen β‐chitin was established: deproteinisation with 3% NaOH for 30 min at 15 psi/121 °C and a solid/solvent ratio of 1:10 (w/v) and a subsequent demineralisation with 1 N HCl for 30 min at room temperature and a solid/solvent ratio of 1:10 (w/v). Squid pen β‐chitin contained 6.29% nitrogen, 0.25% ash, and negligible fat with degree of acetylation of 94.02%, residual amino acid of 0.499 g/100 g and bulk density of 0.28 g mL^?1. Depending on its particle size, squid pen β‐chitin visually looked white (L* = 82.82, a* = ?0.67, b* = 6.31; particle size of 0.15–0.18 mm) or light grey (L* = 62.88, a* = 0.33, b* = 10.66; particle size of 0.425–0.841 mm). Water, fat and dye‐binding capacity of squid pen β‐chitin was 694.67%, 194.03% and 79.81%, respectively.     

Improvement of L(+)‐lactic acid production from cassava wastewater by Lactobacillus rhamnosus B 103

BACKGROUND: L (+)‐Lactic acid is used in the pharmaceutical, textile and food industries as well as in the synthesis of biodegradable plastics. The aim of this study was to investigate the effects of different medium components added in cassava wastewater for the production of L (+)‐lactic acid by Lactobacillus rhamnosus B 103. RESULTS: The use of cassava wastewater (50 g L^?1 of reducing sugar) with Tween 80 and corn steep liquor, at concentrations (v/v) of 1.27 mL L^?1 and 65.4 mL L^?1 respectively led to a lactic acid concentration of 41.65 g L^?1 after 48 h of fermentation. The maximum lactic acid concentration produced in the reactor after 36 h of fermentation was 39.00 g L^?1 using the same medium, but the pH was controlled by addition of 10 mol L^?1 NaOH. CONCLUSION: The use of cassava wastewater for cultivation of L. rhamnosus is feasible, with a considerable production of lactic acid. Furthermore, it is an innovative proposal, as no references were found in the scientific literature on the use of this substrate for lactic acid production. Copyright © 2010 Society of Chemical Industry     

Comparison of the performances of different fermentation strategies on cell growth and bacteriocin production by Lactobacillus curvatus CWBI‐B28

The dynamics of cell growth and bacteriocin production by Lactobacillus curvatus CWBI‐B28 in modified De Man/Rogosa/Sharp (mMRS) broth with various concentrations of glucose and complex nitrogen source (CNS; peptone, yeast extract and meat extract) was investigated in flask fermentations and in a laboratory fermentor using batch and fed‐batch cultivations. In fed‐batch fermentation the rate of feeding of the reactor with the substrates was either maintained constant (0.12 L h^?1) or varied exponentially as a function of time. The results showed that both cell growth and bacteriocin activity were influenced by changes in the concentrations of glucose and CNS. Optimal growth and bacteriocin activity were obtained in mMRS broth containing 40 g L^?1 glucose and 40 g L^?1 CNS (mMRS_40/40). A bacteriocin titre of 4266 AU mL^?1 and a cell count of 8.7 log colony‐forming units (cfu) mL^?1 were recorded when this medium was used for cultivation. In batch fermentation using the same medium, a higher cell count (9.5 log cfu mL^?1) and twice as much bacteriocin as in flask fermentation were produced. The highest bacteriocin titre (8533 AU mL^?1) was obtained with fed‐batch fermentation at an exponentially varying rate of feeding. Bacteriocin activity and cell dry mass did not always correlate. Copyright © 2007 Society of Chemical Industry     

Regulation of thiamine synthesis in Saccharomyces cerevisiae for improved pyruvate production

Metabolic engineering of Saccharomyces cerevisiae for high‐yield production of carboxylic acid requires a cytosolic pyruvate pool as precursor. In this study, a novel strategy to improve pyruvate production and reduce metabolic by‐products via regulating thiamine synthesis was explored. Two of the thiamine biosynthesis regulatory genes, THI2 and THI3, were disrupted in the S. cerevisiae parent strain FMME‐002. The mutants FMME‐002ΔTHI2 and FMME‐002ΔTHI3 both exhibited an enhanced pyruvate yield. Moreover, FMME‐002ΔTHI2 achieved a relatively higher pyruvate production, and the highest concentration of pyruvate was achieved when 0.04 µ m thiamine was added. Enzyme assays and fermentation profiles of the THI2‐complemented strain indicated that the observed metabolic changes represented intrinsic effects of THI2 deletion on the physiology of S. cerevisiae. Under optimal C:N ratio conditions, FMME‐002ΔTHI2 produced pyruvate up to 8.21 ± 0.30 g/l, whereas the ethanol titre decreased to 2.21 ± 0.24 g/l after 96 h of cultivation. These results demonstrate the possibility of improving pyruvate production by regulating thiamine synthesis in S. cerevisiae. Copyright © 2012 John Wiley & Sons, Ltd.     

响应面法优化丙酮酸发酵培养基

采用响应面法(RSM)对丙酮酸发酵培养基成分葡萄糖、硫酸铵、蛋白胨进行优化,采用多元二次回归方程拟合3种因素与丙酮酸含量间的函数关系,并得到了最佳条件。在优化培养条件下,发酵液中丙酮酸的浓度由35.4g/L提高到41.57g/L,在5L罐的最佳浓度下丙酮酸产量71.23g/L比原产量65.76g/L提高8.3%。     

Bistage control of pH for improving exopolysaccharide production from mycelia of Ganoderma lucidum in an air-lift fermentor

It was found that pH control definitely affects mycelial cell growth and exopolysaccharide (EPS) production of the mycelial cultivation of Ganoderma lucidum. Compared to the case of uncontrolled pH cultivation, a culture system whose pH was kept constant at 3 and 6 exhibited improved mycelial cell growth and EPS production, respectively. The bistage pH control technique, that is, shifting the pH from 3 to 6 at the initial phase of the exponential growth, is introduced to improve cell growth and EPS production. This technique can greatly increase EPS production to 20.1 g/l from 4.1 g/l in the case of uncontrolled pH cultivation, without adverse effects on cell growth as in the case of constant maintenance of a high pH. It was also proved that bistage pH control retained the desirable morphologies of the mycelia during cultivation and resulted in low viscosity and yield stress of the culture broth. It will be useful for the application of the culture process to mycelial growth in a large-scale fermentor.     

Effects of NaCl, lactose and availability of oxygen on tyramine production by the Enterococcus durans CCDM 53

Combined effects of concentration of lactose (5?g/L), NaCl (20?g/L) and aero/anaerobiosis on production of tyramine by Enterococcus durans CCDM 53 were subjected to a study. The influence of the above factors and temperature of cultivation (10?±?1?°C) was monitored under conditions applied in real technological processes of cheese production; the enterococci act as non-starter lactic acid bacteria. Production of tyramine by E. durans CCDM 53 was mainly influenced by both concentration of NaCl in cultivation medium and presence/absence of oxygen in the environment. The highest production of tyramine occurred during cultivation under anaerobic conditions in the presence of the highest (20?g/L) applied concentration of NaCl and lactose (5?g/L). In the media with equal concentrations of NaCl and lactose, the concentrations of tyramine grew higher under anaerobic conditions than in aerobic environment. Regarding cultivation media with various levels of NaCl and lactose, higher production of tyramine was always found in the anaerobic environment.     

Addition of Rifampicin to Bolton Broth to Inhibit Extended‐Spectrum β‐Lactamase‐Producing Escherichia coli for the Detection of Campylobacter

Exponential growth of extended‐spectrum β‐lactamase (ESBL)‐producing Escherichia coli in Campylobacter media has become a common problem for the detection of Campylobacter in chicken meats. We investigated the minimum inhibitory concentration of 40 ESBL‐producing E. coli isolates from meats obtained from various countries against antibacterial agents in Bolton broth (cefoperazone, vancomycin, and trimethoprim). All ESBL‐producing E. coli strains were resistant to cefoperazone and vancomycin, whereas 50% of them were resistant to trimethoprim and grew in Bolton broth. We found that 20 μg/mL of rifampicin inhibited the growth of trimethoprim‐resistant E. coli strains. Hence, we added 20 μg/mL of rifampicin to Bolton broth to improve the isolation of Campylobacter from chicken carcass rinses. The isolation rate of Campylobacter was significantly higher in the modified broth (44 out of 58, 75.9%, P < 0.05) than in the normal broth (0 out of 58, 0%). Furthermore, the number of agar plates with non‐Campylobacter spp. was much lower after enrichment in the modified broth (4 out of 58, 6.9%, P < 0.05) than in the normal broth (58 out of 58, 100%).     

Fermented sugarcane and pineapple beverage produced using Saccharomyces cerevisiae and non‐Saccharomyces yeast

The potential of Saccharomyces cerevisiae (strains UFLA CA11 and UFLA FW15) and Pichia caribbica (UFLA CAF733) to produce a fermented sugarcane and pineapple drink was evaluated. Co‐ and pure cultures using different proportions of sugarcane juice and pineapple pulp (80:20, 70:30 and 60:40) were prepared. The sugar concentration of the must was adjusted to 16° Brix and was inoculated with approximately 7 log CFU/mL. After a preliminary test and based on higher concentrations of desirable volatile components, low production of acetic acid, high production of ethanol, and kinetic parameters, P. caribbica was chosen to perform the fermentation in 5 L batches. The fermentation performed with P. caribbica in the proportion of 60:40 showed a yield in ethanol of 0.45 g/g, an ethanol productivity of 1.32 g/L/h and a fermentation efficiency of 88.22%. The maximum ethanol concentration was 79.78 g/L and P. caribbica increased concentrations of desirable volatile compounds, such as 2‐phenyethanol, 2‐methyl‐1‐propanol, 3‐methyl‐1‐butanol, ethyl acetate and phenylethyl acetate. Copyright © 2015 The Institute of Brewing & Distilling     

当量生物素控制对谷氨酸棒杆菌发酵产L-异亮氨酸的影响

以谷氨酸棒杆菌YILM 1504为出发菌,研究了生物素对L-异亮氨酸产量的影响。基于多梯度生物素对比实验及中后期生物素外源添加实验,确定了发酵液中最佳生物素浓度及补料工艺。结果显示:在低浓度生物素发酵液中,最适生物素浓度为45μg/L,此时产酸达到42.5g/L,糖酸转化率达到最高,为13.4%;在大于60μg/L高浓度生物素发酵液中,产酸最高为22g/L,表明高浓度生物素不利于产酸发酵。在5L发酵罐中,初始生物素浓度为30μg/L,18,32,42h分别添加10g/L玉米浆(15μg/L生物素),54h产酸量达到了44.5g/L,比初始生物素浓度为30μg/L,后期不补料产酸量提高了49.8%。     

KINETICS OF BIOETHANOL PRODUCTION FROM WHEAT MILLING BY-PRODUCTS

An overview of the potential application of wheat milling by‐products as substrate for bioethanol production is presented. In order to select a suitable microorganism, model fermentations were conducted using glucose and dry baker's yeast. The overall ethanol yield was nearly stable (ca. 0.35 g/g), independent of mash glucose concentration; mashes with 100 g glucose/L resulted in an overall ethanol productivity of 3.48 g/L·h. Slurries containing low‐grade wheat flour (LG) (100, 200 or 300 g/L) were used for simultaneous saccharification and fermentation (SSF) with Zymomonas mobilis. Fermentation performance was evaluated based on ethanol concentration (P), productivity (Q_v), yield (Y_P/S), production rate (Q_p) and glucose consumption rate (Q_s). Mashes containing 200 g LG/L produced about 52 g ethanol/L, with Q_vof 2.17 g/L·h. Based on the relatively high fermentation rate of LG, reaching peak ethanol productivity within ca. 9 h of SSF, considerable savings on fermentation time was achieved. Using Z. mobilis for LG fermentation, P was about 30% higher than that obtained with Saccharomyces cerevisiae.     

LACTOBACILLUS CURVATUS PRODUCES A BACTERIOCIN-LIKE AGENT ACTIVE AGAINST GRAM-NEGATIVE PATHOGENIC BACTERIA

Isolates from fermented foods were screened for antimicrobial activity against gram‐negative bacteria. The most active isolate was identified as a Lactobacillus curvatus by biochemical analysis and ribotyping, and the isolate was designated as OSY‐HJC6. Lactobacillus curvatus OSY‐HJC6 was further tested for intracellular and extracellular production of antimicrobial agents. A reduction of > 8 log₁₀ cfu/mL was observed when cell suspensions of Salmonella enterica serovar Enteritidis and Escherichia coli O157:H7 were treated with equal volumes of Lb. curvatus culture supernatant. Gram‐positive bacteria were not sensitive to the culture supernatant, but antimicrobial activity was detected when the cell extract was tested against several gram‐positive and gram‐negative bacteria. Culture supernatant and cell extract retained the antimicrobial activity after heating at 60–100C for 10 min but not after protease treatment. The cell extract of Lb. curvatus retarded the growth of E. coli p220 in broth medium and food extracts (i.e., bacteriostatic action) but showed bactericidal activity against the bacterium in phosphate buffer.     

Fed‐batch cultures of phaffia rhodozyma in xylose‐containing media made from wood hydrolysates

Abstract

Eucalyptus wood samples were treated with 3% aqueous sulphuric acid to obtain xylose‐containing solutions. The liquors from treatments were neutralized, contacted with charcoal and supplemented with nutrients to obtain culture media suitable for proliferating the carotenoid‐producing yeast, Phaffia rhodozyma NRRL Y‐17268. Two fed‐batch strategies (with continuous or intermittent feeding) were assayed as operational procedures for improving the carotenoid production. Biomass concentration of 10.3 g cells/L and volumetric pigment concentration of 8.15 mg total carotenoids/L (with 7.19 mg astaxanthin/L) were reached with continuous feeding, whereas improved results (30.6 g cells/L and 33.5 mg total carotenoids/L with 30.5 mg astaxanthin/L) were obtained with intermittent feeding.     

Effects of culture substrates on taste component content and taste quality of Lentinula edodes

In this research, the effects of culture substrates on taste component content of Lentinula edodes were studied, and the resulting taste quality of L. edodes was evaluated. The results revealed that single‐carbon and single‐nitrogen sources were beneficial to production of soluble sugars and polyols, organic acids and sweet amino acids, whereas a single‐carbon source was beneficial to essential amino acid production, and a mixed‐carbon source was beneficial to umami 5′‐nucleotides and high mushroom production yield. High C/N values were beneficial to trehalose, arabitol, malic acid, and succinic acid production, while low C/N values were beneficial to mannitol and citric acid production. L. edodes fruiting bodies, harvested from a culture substrate containing single carbon, high proportion of cereal bran, had a more palatable taste quality, while a substrate containing bagasse and low C/N values was not suitable for L. edodes cultivation due to unfavourable taste quality. These results provide cultivation information how to obtain fruiting bodies L. edodes with more taste components.     

Identification and characterization of Clostridium paraputrificum M-21, a chitinolytic, mesophilic and hydrogen-producing bacterium

A strictly anaerobic, mesophilic and chitinolytic bacterial strain, M-21, was isolated from a soil sample collected from Mie University campus and identified as Clostridium paraputrificum based on morphological and physiological characteristics, and 16S rRNA sequence analysis. C. paraputrificum M-21 utilized chitin and N-acetyl- -glucosamine (GlcNAc), a constituent monosaccharide of chitin, to produce a large amount of gas along with acetic acid and propionic acid as major fermentation products. Hydrogen and carbon dioxide accounted for 65% and 35% of the gas evolved, respectively. The conditions for 1 l batch culture of C. paraputrificum, including pH of the medium, incubation temperature and agitation speed, were optimized for hydrogen production with GlcNAc as the carbon source. The bacterium grew rapidly on GlcNAc with a doubling time of around 30 min, and produced hydrogen gas with a yield of 1.9 mol H₂/mol GlcNAc under the following cultivation conditions: initial medium pH of 6.5, incubation temperature of 45°C, agitation speed of 250 rpm, and working volume of 50% of the fermentor. The dry cell weight harvested from this culture was 2.0 g/l.