Association of bacteria with the fungal fermentation of soybean tempe

Bacteria grew to viable populations of 10(8)-10(9) cfu/g during the fermentation of soybeans into tempe with the fungus, Rhizopus oligosporus. Bacillus pumilus and B. brevis were the predominant bacterial species, reaching populations of approximately 10(8) cfu/g during the 48 h fermentation. Species of Streptococcus faecium, Lactobacillus casei, Klebsiella pneumoniae and Enterobacter cloacae also contributed to the fermentation and achieved populations of 10(6)-10(7) cfu/g.     

Formation of vitamins by pure cultures of tempe moulds and bacteria during the tempe solid substrate fermentation

The formation of water soluble vitamins (vitamin B₁₂, vitamin B₆, riboflavin, thiamine, nicotinic acid and nicotinamide) during the tempe solid substrate fermentation was investigated. The role of several strains of Rhizopus oligosporus, R. arrhizus , and R. stolonifer and the role of several bacteria in the vitamin formation process were checked. All fungal and bacterial strains were isolated from Indonesian tempe and soaking water samples. The Rhizopus strains formed riboflavin, nicotinic acid, nicotinamide and vitamin B₆. The final concentrations of these substances depended on the different strains involved and on the fermentation time. Isolates of R. oligosporus were generally the best vitamin formers. The moulds did not produce physiologically active vitamin B₁₂. The thiamine content decreased during fermentation. The addition of bacteria, which had been selected in a screening for vitamin B₁₂ production, resulted in an increase of physiologically active vitamin B₁₂. Citrobacter freundii and Klebsiella pneumoniae showed the best formation capabilities. Furthermore, the bacteria produced riboflavin and vitamin B₆ in addition to the moulds. The influence of Rhizopus on the vitamin B₁₂ formation of Cit. freundii was also investigated. The vitamin content of tempe that was fermented with the mould and the bacterium was three times as high as a control fermentation with Cit. freundii only.     

Formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from tempe

Five tempe-derived bacterial strains identified as Micrococcus or Arthrobacter species were shown to transform the soybean isoflavones daidzein and glycitein to polyhydroxylated isoflavones by different hydroxylation reactions. All strains converted glycitein and daidzein to 6,7,4′-trihydroxyisoflavone (factor 2) and the latter substrate also to 7,8,4′-trihydroxyisoflavone. Three strains transformed daidzein to 7,8,3′,4′-tetrahydroxyisoflavone and 6,7,3′,4′-tetrahydroxyisoflavone. In addition, two strains formed 6,7,8,4′-tetrahydroxyisoflavone from daidzein. Conversion of glycitein by these two strains led to the formation of factor 2 and 6,7,3′,4′-tetrahydroxyisoflavone. The structures of these transformation products were elucidated by spectroscopic techniques and chemical degradation. Revision received: 9 September 1995 / Accepted: 21 September 1995     

传统豆酱发酵过程中细菌多样性动态

细菌在豆酱发酵过程中起到非常重要的作用,并与豆酱的风味和质量密切相关,因此研究豆酱中细菌的多样性具有重要意义。以自然发酵的豆酱样品为研究对象,采用细菌16S rDNA的部分可变区的PCR-DGGE技术对自然发酵豆酱样品的细菌群落组成和优势菌群进行研究。结果表明,传统豆酱发酵过程细菌群体中既有原始种群的减少和增长,也有次级种群的增多和演变。在整个发酵过程中,初期和末期以不可培养细菌为主,初期细菌群体快速演替,细菌种群多样性指数在发酵42 d和56 d达到两次高峰。     

Real-time monitoring of the accretion of Rhizopus oligosporus biomass during the solid-substrate tempe fermentation

We describe a novel method for the real-time estimation of the accretion of blomass during the solid-substrate tempe fermentation of soy beans, lupins and quinoa by Rhizopus oligosporus Salto. The method is based on measurements of the dielectric permittivity at radio-frequencies, using a four-terminal instrument (the Bugmeter). In all cases, excellent Ilnearity is observed during the growth phase between the dielectric permittivity and the hyphal length as determined microscopically.     

Pectinolytic activity of bacteria isolated from soil and two fungal strains during submerged fermentation

Seven different strains were selected for their ability to degrade citrus pectin. Alkaline pectinases were produced by five bacterial soil isolates, whereas two fungal strains produced pectinase in an acidic environment. The bacteria were isolated from soil of a plum orchard in Northern Ireland. These isolates produced significant amounts of pectin lyase (PL) and polygalacturonase (PG) with maximum activities of 30.1 and 29.1 U/ml respectively. Fungal strains Aspergillus sp. and PN-1 produced four different pectinolytic activities; endo-PG, exo-PG, pectin esterase (PE) and PL. The Aspergillus sp. produced higher amounts of pectinase than PN-1. The Aspergillus sp. excreted highly stable pectinases, which may be of importance for industrial applications.     

Genetically modified soybeans: false-positive detection in fermented natural soybean (tempe)

Tempe was prepared using mixtures of natural soybean and genetically modified Roundup Ready (RUR) soybean fermented with natural Rhizopus sp. The amount of RUR soybean was quantified using an ELISA plate test. The RUR signal decreased during fermentation. In the control experiments on fermentation of non-RUR soybean, the tempe gave a false-positive RUR signal. The cross-reacting substance was generated only in non-RUR soybean during fermentation by Rhizopus sp., Rhizopus oligosporus, R. oryzae, Mucor rouxii and Aspergillus awamori.     

Nutrition of tempe moulds

The nutritional requirements in vitro of Amylomyces rouxii NRRL 3160, Rhizopus arrhizus NRRL 1469, R. arrhizus NRRL 1526, R. oligosporus NRRL 2710, R. oligosporus NRRL 5905, R. oligosporus NRRL A-10457, R. oryzae IMI 215407, R. oryzae NRRL 3563 and R. stolonifera NRRL A-2293 were investigated. All strains grew in glucose-ammonium-salts medium without addition of vitamins. None utilized, as sole carbon and energy source, raffinose or stachyose, the main flatulence-associated oligosaccharides in soybean. All the strains, except Rhizopus oryzae IMI 215407, utilized one or more long chain fatty acids as sole sources of carbon and energy but only the R. arrhizus strains, R. oryzae NRRL 3563 and R. stolonifera , were able to use glycerol as sole carbon and energy source. Phytic acid was used neither as a source of carbon nor of phosphate by any strain.     

Non-target impacts of soybean rust fungicides on the fungal entomopathogens of soybean aphid

Soybean aphid, Aphis glycines, has caused serious economic damage to soybean across the North Central US since its introduction to North America in 2000. The management of another invasive soybean pest, Asian soybean rust, Phakopsora pachyrhizi, using foliar fungicide applications has the potential to impact soybean aphid populations by suppressing beneficial fungal entomopathogens. In 2005 and 2006, we applied recommended soybean rust fungicide treatments, consisting of strobilurin and triazole fungicides, to small soybean plots in two locations to assess if such applications might suppress aphid fungal epizootics. In Lamberton, MN, in 2005, during the epizootic, fungicide-treated plots averaged 2.0 ± 0.7% (mean ± SE) disease prevalence while untreated plots averaged 14.2 ± 5.6%. In 2007, we applied strobilurin and strobilurin-triazole mix fungicides to single-plant microplots either before or after release of Pandora neoaphidis, the most commonly observed aphid pathogen in 2005 and 2006. Treatments that contained a mixture of two active ingredients significantly lowered peak and cumulative aphid disease prevalence in both early and late reproductive stage soybeans indicating that fungicide mixtures used to manage soybean rust can negatively impact an aphid-specific fungal pathogen. However, no consistent soybean aphid population response was observed in these studies of low levels of aphid fungal infection.     

Unusual bacteria from fungal material

Studies on mycorrhizal fungi of the genera Lactarius and Laccaria revealed bacteria, apparently present within the tissue of the sporocarps. Culture of these bacteria indicated that the predominant strains were of unusual colonial morphology. 10 strains were chosen for further study; these could be grown on plates of nitrogen-free medium for a number of subcultures, but did not fix nitrogen, using the acetylene reduction test. They produced copious amounts of extracellular polysaccharide of varying chemotypes.     

Association of methanogenic bacteria with rumen ciliates

In 11 species of rumen ciliates belonging to nine genera of the family Ophryoscolecidae (order Entodiniomorphida) an ectosymbiosis with methanogenic bacteria was found. The bacteria could be identified as methanogens on the basis of the presence of specific fluorescent coenzymes (F350 and F420). This somatic interaction may reflect a metabolic interaction in which efficient interspecies hydrogen transfer benefits both partners.     

Association of methanogenic bacteria with rumen protozoa

Methanogenic bacteria superficially associated with rumen entodiniomorphid protozoa were observed by fluorescence microscopy. A protozoal suspension separated from strained rumen fluid (SRF) by gravity sedimentation exhibited a rate of methane production six times greater (per millilitre) than SRF. The number of protozoa (per millilitre) in the protozoal suspension was three times greater than that of SRF; however, the urease activity of this fraction was half that of SRF. The methanogenic activity of SRF and the discrete fractions obtained by sedimentation of protozoa correlated with the numbers of protozoa per millilitre in each fraction. Gravity-sedimented protozoa, washed four times with cell-free rumen fluid, retained 67-71% of the recoverable methanogenic activity. Thus it is evident from our observations that many methanogens adhere to protozoa and that the protozoa support methanogenic activity of the attached methanogens. When protozoa-free sheep were inoculated with rumen contents containing a complex population of protozoa, methanogenic activity of the microflora in SRF samples was not significantly enhanced.     

以豆浆为培养基的三种药食用真菌发酵过程多糖变化的研究

为研究豆浆对药食用真菌发酵产多糖的影响,对灵芝、金耳、冬虫夏草三种真菌以豆浆为培养基得到的多糖进行了结构和离子交换层析分析。研究结果表明,三种药食用真菌多糖的结构和性质具有较大差异,在单糖组成方面,葡萄糖、阿拉伯糖和半乳糖三种主要单糖的摩尔百分比具有较大差异,同时多糖中不同分子量的各个组分比例也有较大差异。红外光谱图分析结果显示,金耳和灵芝发酵液较虫草发酵液,多糖峰型变化较大,但二者峰型较相似。三种真菌多糖经离子交换柱分离纯化后发现,三种多糖都是由中性和酸性多糖组成,但冬虫夏草发酵液中的中性多糖比例远低于酸性多糖,而金耳和灵芝多糖中酸性多糖比例高于中性多糖。本研究以豆浆为培养基探究三种药食用真菌发酵多糖结构的变化,不仅为多糖结构研究提供理论支持,还为真菌豆浆发酵产品的开发奠定良好的实践基础。     

Solid state fermentation for the production of lipolytic fungal enzymes

The production of lypolitic moulds in solid state fermentation was studied.Aspergillus oryzae andRhizopus oryzae were grown on rice hulls using different media. Tween 80 and olive oil were employed as main carbon sources; the whole solid cultures were lyophilised and employed in heptane for catalysing the formation of various geranyl esters with molar conversions ranging from 40 to 95%, under optimised conditions, starting from 50 mM geraniol and equimolar amount of the acid.     

Modelling fungal solid-state fermentation: the role of inactivation kinetics

The theoretical mathematical models described in this paper are used to evaluate the effects of fungal biomass inactivation kinetics on a non-isothermal tray solid-state fermentation (SSF). The inactivation kinetics, derived from previously reported experiments done under isothermal conditions and using glucosamine content to represent the amount of biomass, are described in different ways leading to four models. The model predictions show only significant effects of inactivation kinetics on temperature and biomass patterns in the tray SSF after long fermentation periods. The models in which inactivation is triggered by low specific growth rates can predict restricted biomass evolution in combination with a fast temperature increase followed by a slower temperature decrease. Such inactivation might occur when substrate is limiting or products are formed in toxic concentrations. Temperature is predicted to be the key parameter. Oxygen concentration is predicted to become limiting only at high heat conduction and low oxygen diffusion rates. Desiccation of the substrate is predicted not to occur.     

Association of methanogenic bacteria with rumen ciliates.

In 11 species of rumen ciliates belonging to nine genera of the family Ophryoscolecidae (order Entodiniomorphida) an ectosymbiosis with methanogenic bacteria was found. The bacteria could be identified as methanogens on the basis of the presence of specific fluorescent coenzymes (F350 and F420). This somatic interaction may reflect a metabolic interaction in which efficient interspecies hydrogen transfer benefits both partners.     

Assessment of hyphal binding in tempe

An inverse three-point bending test method is described for measuring the strength of the hyphae binding the cotyledons together in soybean tempe. The force to failure and the work to failure both increased from zero values before hyphal development to maximum values after about 30 h incubation and then declined as the tempe aged and hyphae started to senesce. The changes in force to failure and work to failure closely paralleled changes in the appearance and manual assessments of the texture of the tempes.     

Rheological characterization of a fungal fermentation for the production of pneumocandins

The filamentous fungus Glarea lozoyensis produces a novel, pharmaceutically important pneumocandin (B₀) that is used to synthesize a polypeptide, which demonstrates fungicidal activity against clinically relevant pathogens. The scale-up of the pneumocandin fermentation requires an understanding of the rheological properties of the broth and the factors that influence flow behavior. A systematic approach for characterizing the rheological properties using a standard methodology is presented here. An appropriate rheometer was chosen and the effects of shear rate ramping, broth handling, creep and yield testing were examined. The fed-batch fermentation used a soluble production medium that allowed the relationship between biomass and rheological properties to be studied up to the 19-m³ scale. The morphologically heterogeneous broth demonstrated time-dependent shear thinning behavior with thixotropy and a yield stress. The flow curves were described by the power law model, with flow behavior of 0.35-0.4 and consistency index up to 10 Pa.sⁿ. The use of a cup and bob rheometer was preferable to alternative techniques, including turbine and spindle systems defined by Mitschka's technique. The consistency index and flow behavior were shown to have a non-linear relationship with biomass concentrations up to 140 g/L. The consistency index continually increased with biomass during the fermentation, while the flow behavior initially decreased rapidly and then remained at low values for the remainder of the batch cultivation. The consistency index and yield stress were influenced by temperature, osmotic pressure, and pH, while the flow behavior remained independent of these factors.