A differential medium for lactic acid-producing bacteria in a mixed culture

Aims:  Modified deMan-Rogosa Sharpe agar containing bromophenol blue (mMRS-BPB) was tested as a medium for counting and differentiation of each lactic acid-producing bacterium (LAB), especially in a mixed culture.
Methods and Results:  Type strains of 10 LAB species ( Lactobacillus acidophilus , L. brevis , L. bulgaricus , L. gasseri , L. paracasei , L. plantarum , L. reuteri , Weissella confusa , Bifidobacterium bifidum and B. infantis ) and five commercial yogurts were inoculated on plate count agar with bromocresol purple, mMRS, and mMRS-BPB. Each type strain showed more clearly formed colonies on the three media under anaerobic conditions than under aerobic conditions. Especially each type strain produced colonies with specific characteristics of each species on mMRS-BPB. Commercial yogurts produced the largest number of colonies with various shapes and colours on mMRS-BPB.
Conclusions:  Modified deMan-Rogosa Sharpe agar containing bromophenol blue under anaerobic conditions is appropriate for counting and differentiating each LAB in a mixed culture.
Significance and Impact of the Study:  Modified deMan-Rogosa Sharpe agar containing bromophenol blue will be useful in isolation and enumeration of each LAB from fermented foods as well as intestinal microflora.     

Isolation of bovine intestinal Lactobacillus plantarum and Pediococcus acidilactici with inhibitory activity against Escherichia coli O157 and F5

Aims:  The growth rate of bovine lactic acid bacteria (LAB) in five different culture conditions, and their inhibitory activity against Escherichia coli O157 and F5 in two assays was assessed to identify LAB for potential prophylactic use in cattle.
Methods and Results:  106 bovine-derived faecal/intestinal LAB were tested in vitro for tolerance to pH 2·0, pH 4·0, 0·15% and 0·3% bile, aerobic incubation, and for inhibitory activity against E. coli O157 ( n  = 3) and F5 ( n  = 1). While no LAB grew at pH 2·0, LAB survivability varied between 35% and 100% on the other tests. Exactly 7·6% (8/106) of LAB supernatants inhibited the growth of E. coli in two assays, whereas 6·6% (7/106) of isolates enhanced the growth of all E. coli strains. Partial 16s rRNA gene sequencing of six best isolates (95th percentile) revealed that five were Lactobacillus plantarum and one Pediococcus acidilactici.
Conclusion:  Lactobacillus plantarum with acid/bile and aerobic resistance and inhibitory activity against E. coli O157 and F5 inhabit the intestinal tract of healthy cattle. Some LAB may enhance E. coli growth.
Significance and Impact of the Study:  Lactobacillus plantarum and P. acidilactici are natural plant micro-organisms and studied silage inoculants. Their identification from gastrointestinal samples of healthy cattle is prophylactically promising.     

Isolation, characterization and identification of lactic acid bacteria and yeasts from sour Mifen, a traditional fermented rice noodle from China

Aims:  Considering the effect of natural fermentation on the textural improvement of fermented rice noodles in China and South Asia, and given the lack of reports concerning microbial populations and structure in the fermentation process, this study aims to determine the number of viable micro-organisms and identify the species isolated from the local factories, and to assess their potential use as a starter culture from their enzymatic profiles.
Methods and Results:  Fourteen samples from three local factories were analysed for the presence of micro-organisms. A total of 170 lactic acid bacteria (LAB) and 96 yeasts were isolated from the factories. The isolates were phenotypically characterized by using API 50 CHL kits, API 20 Strep kits, API ID 32 C kits and by performing additional biochemical tests. The enzymatic profiles of isolates were assessed by using API ZYM kits. Lactobacillus plantarum and Saccharomyces cerevisiae were identified as predominant species in the fermented supernatants. A majority of the isolates of LAB and yeasts displayed activities of α-glucosidase, β-glucosidase, lipase and trypsin.
Conclusions:  The microbial composition and strain characteristics present in the fermentation supernatant demonstrate that a majority of micro-organisms have the ability to digest starch, sugar, protein or lipid. It supports our previous work in which the rice starch was modified and purified by fermentation and thus improves the texture of rice noodles.
Significance and Impact of the Study:  The dominant strains would be important in developing a starter culture. The results can form the basis for the improvement of product quality and consistency.     

偶氮染料的微生物脱色研究进展

微生物法是染料废水治理的重要方法。本文综述了特异性酶作用下好氧细菌和真菌对偶氮染料的脱色以及厌氧条件下氧化还原介质作为电子穿梭体时偶氮染料的非特异性还原过程。指出厌氧偶氮还原是偶氮染料还原的主要形式, 电子供体不同脱色效率不同。对目前生物法去除偶氮染料存在的问题进行了分析, 提出了相应的对策措施。     

Brevibacillus laterosporus MTCC 2298: a potential azo dye degrader

Aims:  To evaluate the potential of Brevibacillus laterosporus MTCC 2298 for the decolourization of different textile azo dyes including methyl red, mechanism of biotransformation and the toxicity of products.
Methods and Results:  Brevibacillus laterosporus showed decolourization of thirteen different azo dyes including methyl red. Decolourization of methyl red was faster (93% within 12 h) under static condition at the concentration 0·2 g l⁻¹. Induction in the activities of lignin peroxidase, laccase, aminopyrine N-demethylase, NADH-DCIP reductase and malachite green reductase was observed in the cells obtained after decolourization. Fourier transform infra-red spectral analysis of products indicated conversion of methyl red into secondary aryl amines and nitrosamines, which further transformed into the aromatic nitro compounds. Gas chromatography–mass spectroscopy analysis suggested conversion of methyl red into high molecular weight complex derivatives. The heterocyclic substituted aryl amine ( m / z 281), p -(N,N di formyl)-substituted para -di amino benzene derivative ( m / z 355) and p -di-amino benzene derivative ( m / z 282) are the mainly elected biotransformation products. Microbial and phytotoxicity studies suggested nontoxic nature of the biotransformation products.
Conclusions:  Brevibacillus laterosporus has potential for the decolourization of different textile azo dyes.
Significance and Impact of the Study:  Brevibacillus laterosporus decolourized different azo dyes including methyl red and can be utilized for textile dye decolourization.     

Biodegradation of azo dyes in cocultures of anaerobic granular sludge with aerobic aromatic amine degrading enrichment cultures

A prerequisite for the mineralization (complete biodegradation) of many azo dyes is a combination of reductive and oxidative steps. In this study, the biodegradation of two azo dyes, 4-phenylazophenol (4-PAP) and Mordant Yellow 10 (4-sulfophenylazo-salicylic acid; MY10), was evaluated in batch experiments where anaerobic and aerobic conditions were integrated by exposing anaerobic granular sludge to oxygen. Under these conditions, the azo dyes were reduced, resulting in a temporal accumulation of aromatic amines. 4-Aminophenol (4-AP) and aniline were detected from the reduction of 4-PAP. 5-Aminosalicylic acid (5-ASA) and sulfanilic acid (SA) were detected from the reduction of MY10. Subsequently, aniline was degraded further in the presence of oxygen by the facultative aerobic bacteria present in the anaerobic granular sludge. 5-ASA and SA were also degraded, if inocula from aerobic enrichment cultures were added to the batch experiments. Due to rapid autoxidation of 4-AP, no enrichment culture could be established for this compound. The results of this study indicate that aerobic enrichment cultures developed on aromatic amines combined with oxygen-tolerant anaerobic granular sludge can potentially be used to completely biodegrade azo dyes under integrated anaerobic/aerobic conditions. Received: 16 September 1998 / Received revision: 14 December 1998 / Accepted: 21 December 1998     

Lactic acid bacteria associated with a heat-processed pork product and sources of variation affecting chemical indices of spoilage and sensory characteristics

Aims:  To evaluate the potential for developing a quality index for a Danish modified atmosphere packaged (MAP) heat-processed and naturally contaminated pork meat product stored at 5°C.
Methods and Results:  The composition of the predominating microflora and changes in contents of tyramine, arginine, organic acids and sensory characteristics were analysed. The microflora was predominated by Lactobacillus sakei , Leuconostoc carnosum and Carnobacterium divergens . The presence of each species varied between products and batches resulting in limited usefulness of the concentrations of these bacteria or their metabolites as indices of quality. Furthermore, the three species differed in their metabolic activities as shown by use of a model meat extract. However, when MAP storage of the processed pork product was followed by aerobic storage then acetic acid showed some potential as a chemical indicator of sensory quality.
Conclusion:  Variation in processing parameters and spoilage microbiota limited the usefulness of concentrations of micro-organisms and their metabolites as indices of spoilage for the studied processed MAP pork product.
Significance and Impact of the Study:  The present study contributes to an understanding of the difficulties experienced in developing quality indices to be used in the control of microbial spoilage of processed MAP meat products.     

Decolorization of anthraquinone dye intermediate and its accelerating effect on reduction of azo acid dyes by Sphingomonas xenophaga in anaerobic–aerobic process

Decolorization of 1-aminoanthraquinone-2-sulfonic acid (ASA-2) and its accelerating effect on the reduction of azo acid dyes by Sphingomonas xenophaga QYY were investigated. The study showed that ASA-2 could be efficiently decolorized by strain QYY under aerobic conditions according to the analysis of total organic carbon removal and UV-VIS spectra changes. Moreover, strain QYY was able to reduce azo acid dyes under anaerobic conditions. The effects of various operating conditions such as carbon sources, temperature, and pH on the reduction rate were studied. It was demonstrated that ASA-2 used as a redox mediator could accelerate the reduction process. Consequently the reduction of azo acid dyes mediated by ASA-2 and the decolorization of ASA-2 with strain QYY could be achieved in an anaerobic-aerobic process.     

Identification of Quinoide Redox Mediators That Are Formed during the Degradation of Naphthalene-2-Sulfonate by Sphingomonas xenophaga BN6

During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.     

Biological treatment of acid dyeing wastewater using a sequential anaerobic/aerobic reactor system

The treatment of the wastewater taken from a wool dyeing processing in a wool manufacturing plant was investigated using an anaerobic/aerobic sequential system. The process units consisted of an anaerobic UASB reactor and an aerobic CSTR reactor. Glucose, alkalinity and azo dyes were added to the raw acid dyeing wastewater in order to simulate the dye industry wastewater since the raw wastewater contained low levels of carbon, NaHCO₃ and color through anaerobic/aerobic sequential treatment. The UASB reactor gave COD and color removals of 51–84% and 81–96%, respectively, at a HRT of 17 h. The COD and color removal efficiencies of the UASB/CSTR sequential reactor system were 97–83% and 87–80%, respectively, at a hydraulic retention time (HRTs) of 3.3 days. The aromatic amines (TAA) formed in the anaerobic stage were effectively removed in the aerobic stage.     

Decolorization of water and oil-soluble azo dyes by <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>

The capability of Lactobacillus acidophilus and Lactobacillus fermentum to degrade azo dyes was investigated. The bacteria were incubated under anaerobic conditions in the presence of 6 μg/ml Methyl Red, Ponceau BS, Orange G, Amaranth, Orange II, and Direct Blue 15; 5 μg/ml Sudan I and II; or 1.5 μg/ml Sudan III and IV in deMann–Rogosa–Sharpe broth at 37°C for 36 h, and reduction of the dyes was monitored. Both bacteria were capable of degrading all of the water-soluble azo dyes to some extent. They were also able to completely reduce the oil-soluble diazo dyes Sudan III and IV but were unable to reduce the oil-soluble monoazo dyes Sudan I and II to any significant degree in the concentrations studied. Growth of the bacteria was not significantly affected by the presence of the Sudan azo dyes. Metabolites of the bacterial degradation of Sudan III and IV were isolated and identified by liquid chromatography electrospray ionization tandem mass spectrometry analyses and compared with authentic standards. Aniline and o-toluidine (2-methylaniline), both potentially carcinogenic aromatic amines, were metabolites of Sudan III and IV, respectively.     

Identification of quinoide redox mediators that are formed during the degradation of naphthalene-2-sulfonate by Sphingomonas xenophaga BN6

During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.     

Development of detection medium for hard-to-culture beer-spoilage lactic acid bacteria

Aims:  To develop a detection medium for hard-to-culture beer-spoilage lactic acid bacteria (LAB). Methods and Results:  Four hard-to-culture beer-spoilage strains of LAB, belonging to Lactobacillus paracollinoides and Lactobacillus lindneri, have been obtained by repeatedly subculturing the wild-type strains in beer. To develop a countermeasure against these hard-to-culture beer-spoilage LAB, a beer-based medium was modified. As a consequence, the supplementation of a small amount of de Man Rogosa Sharpe medium was found to enhance the growth of hard-to-culture beer-spoilage LAB strains obtained in this study. In addition, sodium acetate was shown to improve the selectivity of this beer-based medium. Further comparative study was performed with five other media widely used for the detection of beer-spoilage LAB in the brewing industry. This study revealed that the newly developed medium, designated advanced beer-spoiler detection (ABD) medium, possessed superior sensitivity for hard-to-culture beer-spoilage LAB and comparable sensitivity with easy-to-culture beer-spoilage LAB. Moreover, ABD medium was found to suppress the growth of nonspoilage micro-organisms, and thereby allow the selective growth of beer-spoilage LAB. Conclusions:  Advanced beer-spoiler detection medium is considered as an effective tool for comprehensive detection of beer-spoilage LAB in breweries. Significance and Impact of the Study:  The detection by ABD medium can be used as an indicator for differentiating the beer-spoilage ability of LAB without further confirmatory tests in breweries.     

Special traits of decomposition of azo dyes by anaerobic microbial communities

We present the results of an investigation into the special traits of conversion of azo dyes Acid Orange 6, Acid Orange 7, Methyl Orange, and Methyl Red under anaerobic conditions in comparison to aerobic conditions. In the presence of oxygen, only Methyl Red underwent decomposition, while under oxygen-free conditions, all remaining substances were fully decolourised under the action of a methanogenous consortium of microorganisms. The products of reduction of the azo bond are determined in the case of each dye. Introduction of additional acceptors of electrons (sulfate and nitrate) had a negative influence on the discoloration of azo dyes. Addition of ethanol as an available organic cosubstrate accelerated decomposition of azo dyes both under methanogenous and sulfate- and nitrate-reducing conditions. There is no direct correlation between the rates of conversion of azo dyes under anaerobic conditions or their toxicity to acetoclastic methanogens. Changes in the morphological composition of the community decolouring an azo dye depended on the duration of its impact on microorganisms. The mechanism of the reduction of the azo bond under the action of substances acting as mediators is explained. These substances are products of the metabolism of the microbial community in anaerobic conditions. It is shown that the supposed mediators NADH and sulfide efficiently decolourise azo dyes in a cell-free system, while riboflavin significantly increased the rate of conversion of substrates in recurrent cycles of discoloration only in the presence of an anaerobic microbial consortium.     

Effect of starter culture inoculation on feed hygiene and microbial population development in fermented pig feed composed of a cereal grain mix with wet wheat distillers' grain

Aims:  Investigating the influence of an added starter culture on the properties of fermented liquid pig feed.
Methods and Results:  Diets of cereal grain blended with wet wheat distillers' grain that were either not inoculated (WWDG), inoculated with a silage starter culture at start (WWDGsc1) or at start and at each backslopping (replacement of 80% the content with fresh mixture, simulating feed outtake, WWDGsc5) were fermented for 5 days, followed by 5 days of daily backslopping. Numbers of undesirable micro-organisms (enterobacteria, moulds) were reduced in all fermentations; particularly enterobacteria in the starter culture inoculated diets. Lactobacillus plantarum present in the starter culture became dominant in diets WWDGsc1 and WWDGsc5. However, Lactobacillus panis that was dominating WWDG was also abundant in WWDGsc1 and WWDGsc5. Yeast populations were not influenced by the starter culture, with Pichia fermentans dominating all fermentations. All diets had similar chemical characteristics with the exception of a significant increase of all tested organic acids in WWDGsc5.
Conclusions:  The addition of a starter culture influences the bacterial population in fermented liquid feed, but there is also a strong impact of the flora already present in the feed ingredients. The yeast population is not influenced by adding a lactic acid bacteria (LAB) starter culture. A consortium of LAB and yeast strains adapted to the fermentation should be used as starter culture.
Significance and Impact of the Study:  The results suggest that it is possible to influence the current unpredictable and spontaneous process of feed fermentation when appropriate starter cultures are used. For this purpose, LAB and yeasts with desirable characteristics should be isolated.     

氧气对混合菌群脱色降解偶氮染料效果的影响

【背景】偶氮染料及其中间产物具有一定的环境毒性,利用混合菌群降解偶氮染料是一种环境友好型方法,但降解过程中氧气的存在起到至关重要的作用,可以促进或抑制偶氮染料的微生物降解作用。【目的】探讨氧气对偶氮染料微生物脱色液的影响,分析氧气对混合菌群脱色降解偶氮染料效果的影响。【方法】利用混合菌群DDMY1在3种培养条件(好氧、厌氧、兼氧)下,对7种偶氮染料进行脱色降解,探讨偶氮染料脱色液对氧气的响应情况,利用紫外可见分光光度法(ultraviolet visible spectrophotometry,UV-vis)和傅里叶变换红外光谱法(Fourier transform infrared spectroscopy,FTIR)对脱色产物进行分析。【结果】在兼氧和厌氧条件下反应48 h后的染料脱色液,与氧气充分接触后,部分偶氮染料微生物脱色液发生较为明显的复色现象,如活性黑5、直接黑38;UV-vis分析结果表明,这种复色现象是由于脱色液与氧气接触之后产生新物质所致;FTIR分析结果表明,混合菌群对发生复色反应的偶氮染料仍然具有一定脱色降解效果,但是脱色尚不够完全。【结论】兼氧和厌氧条件下,氧气对部分偶氮染料微生物脱色液具有较为明显的影响,从而影响混合菌群对偶氮染料的整体脱色效果,这可为今后研究偶氮染料彻底生物降解提供理论基础。     

Development of a new method for the detection of lactic acid bacteria capable of protecting ham against Enterobacteriaceae

Aims:  Challenge trials seem to be the best assessment approach to evaluate the potency of food protective cultures. However, this method is time consuming and often difficult to implement. Here, we describe the development of the 'sequential culturing method', a new method for the screening of strains as protective cultures.
Methods and Results:  The sequential culturing method is based on the simulation, in a meat simulation medium (named BHI5L200), of the inhibition of Enterobacteriaceae by Lactobacillus , observed previously in situ . Results obtained with this sequential culturing method were in good agreement with those of the challenge test on sliced cooked ham and confirmed the antagonistic potency of Lactobacillus . The results obtained from the screening of 187 lactic acid bacteria (LAB) indicated that Lactobacillus sakei , Lactococcus lactis diacetylactis and Carnobacterium spp. were strong inhibitors of Enterobacteriaceae whereas Pediococcus spp., Leuconostoc spp., Weisselia spp. and other species of Lactobacillus and Lactococcus , did not possess the same inhibitory capacity.
Conclusions:  Sequential culturing method appeared to be a useful tool to rapidly select LAB cultures which are good candidates for bioprotection of meat.
Significance and Impact of the Study:  Sequential culturing method and simulating media could efficiently mimic challenge test experiments in the selection of potential protective culture for all types of food, on the condition to have the appropriate simulating media, corresponding to the food for which protective cultures were searched.     

Characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products

Aims:  To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides.
Method and Results:  Seven LAB strains [ Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121°C for 15 min, stable at pH 2–10, sensitive to protease, insensitive to α-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N -terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins.
Conclusions:  Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics.
Significance and Impact of the Study:  The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori .     

Production of conjugated linoleic acid by probiotic Lactobacillus acidophilus La-5

Aims:  To study the ability of the probiotic culture Lactobacillus acidophilus La-5 to produce conjugated linoleic acid (CLA), which is a potent anti-carcinogenic agent.
Methods and Results:  The conversion of linoleic acid to CLA was studied both by fermentation in a synthetic medium and by incubation of washed cells. Accumulation of CLA was monitored by gas chromatography analysis of the biomass and supernatants. While the fermentation conditions applied may not be optimal to observe CLA production in growing La-5 cells, the total CLA surpassed 50% of the original content in the washed cells after 48 h under both aerobic and micro-aerobic conditions. The restriction of oxygen did not increase the yield, but favoured the formation of trans, trans isomers.
Conclusions:  The capability of L. acidophilus La-5 to produce CLA is not dependant on the presence of milk fat or anaerobic conditions. Regulation of CLA production in this strain needs to be further investigated to exploit the CLA potential in fermented foods.
Significance and Impact of the study:  Knowledge gained through the conditions on the accumulation of CLA would provide further insight into the fermentation of probiotic dairy products. The capacity of the nongrowing cells to produce CLA is also of great relevance for the emerging nonfermented probiotic foods.