Evolutionary engineering to enhance starter culture performance in food fermentations

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The presence of salt and a curing agent reduces bacteriocin production by Lactobacillus sakei CTC 494, a potential starter culture for sausage fermentation

The specific conditions in the batter of raw fermented sausages may reduce the efficiency of bacteriocin-producing starter cultures. In this work, using in vitro fermentation, we found that sodium chloride and sodium nitrite interfere with the growth of Lactobacillus sakei CTC 494, an organism which produces the antilisterial bacteriocin sakacin K. Because sakacin K production follows primary metabolite kinetics, a decrease in cell formation resulted in a decrease in sakacin K production as well. Sodium chloride dramatically influenced bacteriocin production by decreasing both biomass production and specific bacteriocin production. Sodium nitrite, however, had no effect on specific bacteriocin production and decreased bacteriocin production only because of its effect on cell growth. Moreover, sodium nitrite enhanced the toxic effect of lactic acid on bacterial growth.     

Novel Leuconostoc citreum starter culture system for the fermentation of kimchi, a fermented cabbage product

To determine the dominant microorganisms involved in kimchi fermentation and to examine their effect on kimchi fermentation, we randomly isolated and characterized 120 lactic acid bacteria from kimchi during a 5-day fermentation at 15°C. Leuconostoc citreum was dominant during the early and mid-phases of kimchi fermentation whereas Lactobacillus sake/Lactobacillus curvatus or Lactobacillus brevis were found during later stages. Eighty-two out of 120 isolates (68%) were identified as Leuconostoc citreum by means of a polyphasic method, including 16S rDNA sequencing and DNA/DNA hybridization. A few Weissella confusa-like strains were also isolated during the mid-phase of the fermentation. Strain IH22, one of the Leuconostoc citreum isolates from kimchi, was used as an additive to evaluate growth and acid production in kimchi fermentation. This strain was consistently over 95% of the population in IH22-treated kimchi over a 5-day fermentation, while heterogeneous lactic acid bacteria were observed in the control kimchi. The pH in IH22-treated kimchi dropped rapidly but was stably maintained for 5 days, compared to its slow and prolonged decrease in the control kimchi. These results indicate that Leuconostoc citreum IH22 dominates over and retards the growth of other lactic acid bacteria in kimchi, suggesting it can be used as a bacterial starter culture to maintain the quality of kimchi for prolonged periods. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation of the lipolytic activity of starter cultures for meat fermentation purposes

A modified pork fat based agar method was specially developed to determine the lipolytic activity of starter strains used for meat fermentations. The lipolytic ability of strains of lactobacilli, pediococci, staphylococci and micrococci, was examined on agar plates using different substrates and in a lipid broth model system. The screening results indicate that lipase activity was confined to strains of Staphylococcus and Micrococcus . None of the lactic acid bacteria tested showed lipolytic activity. A strain of Staphylococcus xylosus , which displayed high lipolytic activity during the screening process was examined to find its optimum conditions for lipase activity regarding pH, temperature and NaCl concentration. The lipolytic activity of seven bacterial strains was determined under optimum conditions (0% salt, pH 7 and 30 °C) and sausage conditions (3·5% salt, pH 5 and 20 °C) using fat buffer model systems. High lipolytic activity was determined for all seven strains under optimal conditions, whereas no activity was detected under fermented sausage conditions.     

Halobacterium sp. SP1(1) as a starter culture for accelerating fish sauce fermentation

Aims: Application of Halobacterium sp. SP1(1) for the acceleration of fish sauce fermentation. Methods and Results: Traditional fish sauce fermentation was mimicked using Halobacterium sp. SP1(1) as starter culture. Protease activity, peptide release and α‐amino content (parameters used to monitor the progress of the fermentation) were high at day 10 in tests and day 20 in un‐inoculated controls. The total protein and nitrogen contents were also high in tests compared with controls. The amino acid profile observed at the end of fermentation in experimental samples, when compared with the commercial sauce preparation, was found to be better with respect to flavour and aroma contributing amino acids as well as essential amino acid lysine. Microflora analysis of the final fish sauce revealed the absence of any nonhalophilic or halotolerant micro‐organisms. The protease‐producing halophilic isolates obtained from the fish sauce of eviscerated and uneviscerated controls were identified as Halobacterium sp. F1 and F2, respectively, by 16S rDNA sequence analysis. Conclusions: Exogenous augmentation of Halobacterium sp. SP1(1) accelerated the fish sauce fermentation process with an additive effect on the existing natural microflora present in the fish during fermentation. Halobacterium sp SP1(1), therefore, can be used as an important starter culture for accelerating the fish fermentation process, which is attributed to its extracellular protease. Significance and Impact of the Study: The present study is the first report on use of Halobacterium species as a starter culture for accelerating fish sauce fermentation. Use of halobacterial starter cultures may revolutionize the process in fish sauce industries by reducing the fermentation time and making the process more economical with improved nutritive value of product.     

Functional yeast starter cultures for cocoa fermentation

The quest to develop a performant starter culture mixture to be applied in cocoa fermentation processes started in the 20th century, aiming at achieving high-quality, reproducible chocolates with improved organoleptic properties. Since then, different yeasts have been proposed as candidate starter cultures, as this microbial group plays a key role during fermentation of the cocoa pulp-bean mass. Yeast starter culture-initiated fermentation trials have been performed worldwide through the equatorial zone and the effects of yeast inoculation have been analysed as a function of the cocoa variety (Forastero, Trinitario and hybrids) and fermentation method (farm-, small- and micro-scale) through the application of physicochemical, microbiological and chemical techniques. A thorough screening of candidate yeast starter culture strains is sometimes done to obtain the best performing strains to steer the cocoa fermentation process and/or to enhance specific features, such as pectinolysis, ethanol production, citrate assimilation and flavour production. Besides their effects during cocoa fermentation, a significant influence of the starter culture mixture applied is often found on the cocoa liquors and/or chocolates produced thereof. Thus, starter culture-initiated cocoa fermentation processes constitute a suitable strategy to elaborate improved flavourful chocolate products.     

Using a flexible shaft agitator to enhance the rheology of a complex fungal fermentation culture

The rheology behavior of biological fluids particularly when the viscosity is high and rheology is complex, is an important issue to understand, particularly for studies in mass-transfer and for solving technical problems with mixing in stirred bioreactors. In this paper, the use of a Swingstir^® impeller during the fermentation of Aspergillus oryzae resulted in decreases from the parameters of a power-law model, in viscosity and in the thixotropic behavior of a cultivation broth. The results showed that both the K _L a and the alpha amylase activity were improved when using the Swingstir^® in comparison with Fullzone^® impeller (FZ) at the same level of energy consumption. Increasing the pellet porosity during mixing via the Swingstir^® resulted in increases in oxygen mass transfer and the average shear stress.     

The cocoa bean fermentation process: from ecosystem analysis to starter culture development

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro‐organisms, namely yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which results in well‐fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp‐bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa‐derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully.     

Patagonian red wines: selection of Lactobacillus plantarum isolates as potential starter cultures for malolactic fermentation

The aim of this study was to evaluate fifty-three Lactobacillus plantarum isolates obtained from a Patagonian red wine, molecularly identified and typified using RAPD analysis, in order to select starter cultures for malolactic fermentation (MLF). The results obtained suggest a considerable genetic diversity, taking into account that all L. plantarum isolates were obtained from one cellar and one vintage. Based on the capacity to tolerate a concentration of 14 % ethanol in MRS broth for 2 days, eight isolates were selected for the subsequent analysis. The incidence of various wine stress factors (ethanol, acid pH, lysozyme and sulfur dioxide) on isolates growth was studied. Besides, glucosidase and tannase activities were evaluated, and the presence of genes involved in the synthesis of biogenic amines was examined by PCR. A previously characterized indigenous Oenococcus oeni strain was included with comparative purposes. Differences in technologically relevant characteristics were observed among the eight L. plantarum selected isolates, revealing an isolate-dependent behavior. Detectable glucosidase and tannase activities were found in all isolates. The presence of genes encoding histidine and tyrosine descarboxylases and putrescine carbamoyltransferase was not detected. The ability of L. plantarum isolates to grow and consume l-malic acid in simulated laboratory-scale vinifications revealed that two of them could be considered as possible MLF starter cultures for Patagonian red wines. These isolates will be subjected to further analysis, for a final winery technological characterization.     

Inoculum source for anaerobic fermentation of coffee pulp

Summary An experiment was conducted to find out the correct seeding (inoculum) material for the start up of anaerobic digestion using coffee pulp as substrate. Three different inocula viz., slurry from cowdung biogas plant, sewage sludge and rumen fluid were tried for their efficiency in producing maximum methanogenic activities. The result revealed that the sludge from a biogas plant is the best source of inoculum, which enhanced microbial activities, substrate utilization and gas yield. The various physico-chemical changes that occurred during fermentation were also discussed in detail.     

Biotechnological potential of coffee pulp and coffee husk for bioprocesses

Advances in industrial biotechnology offer potential opportunities for economic utilization of agro-industrial residues such as coffee pulp and coffee husk. Coffee pulp or husk is a fibrous mucilagenous material (sub-product) obtained during the processing of coffee cherries by wet or dry process, respectively. Coffee pulp/husk contains some amount of caffeine and tannins, which makes it toxic in nature, resulting the disposal problem. However, it is rich in organic nature, which makes it an ideal substrate for microbial processes for the production of value-added products. Several solutions and alternative uses of the coffee pulp and husk have been attempted. These include as fertilizers, livestock feed, compost, etc. However, these applications utilize only a fraction of available quantity and are not technically very efficient. Attempts have been made to detoxify it for improved application as feed, and to produce several products such as enzymes, organic acids, flavour and aroma compounds, and mushrooms, etc. from coffee pulp/husk. Solid state fermentation has been mostly employed for bioconversion processes. Factorial design experiments offer useful information for the process optimization. This paper reviews the developments on processes and products developed for the value-addition of coffee pulp/husk through the biotechnological means.     

Microflora of banh men,a fermentation starter from Vietnam

In this study Banh mensamples obtained from Vietnam were analysed in terms of pH, moisture content and fungal composition. The banh men pH proved to be acidic with a mean pH of 5.76. Moisture content was 13.6%. Total mould and yeast counts yielded 1.3 × 10⁶ and 4.3 × 10⁶ c.f.u./g-fresh sample, respectively. A total of 53 fungal isolates were obtained from 20 moulds and 33 yeasts. The mould isolates were identified as Rhizopus oryzae, Mucor indicus, Mucor circinilloides, and Amylomyces rouxii. The yeast isolates were identified as Saccharomyces cerevisiae, Hyphopichia burtonii, Saccharomycopsis fibuligera, Pichia anomala, and Candida sp. Based on the parameters used in this study, it can be deduced that banh men is similar to ragi and other Asian fermentation starters.     

Germination of coffee seeds and its significance for coffee quality

Besides genotypic characteristics, the crucial factor that determines coffee quality is the mode of post-harvest treatment, i.e., the wet and dry processing. Up to now, the resulting characteristic flavour differences between these differentially processed coffees were attributed exclusively to differences in starting material. However, as these quality differences are still evident, even when identical coffee samples were processed by the two methods in parallel, the differences must be created by metabolic processes in the coffee beans themselves. Based on expression studies of the germination-specific isocitrate lyase and the resumption of cell cycle activity, monitored by the abundance of beta-tubulin, we evidence that germination is initiated in coffee seeds during the course of standard coffee post-harvest treatments. The extent and nature of the germination processes depend on the processing method. The coherence of metabolic events, substantial differences in the chemical composition of the coffee beans, and the generation of specific coffee qualities establishes the basis for a quite novel approach in coffee research.     

On-line monitoring of important organoleptic methyl-branched aldehydes during batch fermentation of starter culture Staphylococcus xylosus reveal new insight into their production in a model fermentation

A small fermentor (55 mL) was directly interfaced to a membrane inlet mass spectrometer for continuous on-line monitoring of oxygen and volatile metabolites during batch fermentations of the starter culture Staphylococcus xylosus. Using this technique, we were able to correlate production of the very important flavor compounds 2-methylbutanal, 3-methylbutanal, and 2-methylpropanal with various growth conditions. We found that the aldehydes were present in the culture broth only as transient metabolites. They were produced in the exponential growth phase, reached a maximum concentration when the culture became anaerobic, and then they rapidly disappeared from the culture medium. This general pattern was observed for three different strains of S. xylosus and S. carnosus. Small amounts of inoculum or increased exposure to oxygen were found to favor production of the aldehydes as a result of a longer aerobic growth period. Growing S. xylosus under conditions resembling those in a fermented sausage revealed that NaCl (5%) increased aldehyde production considerably, whereas KNO(3) (0.03%) or NaNO(2) (0.03%) had little effect. A lowering of pH from 7.2 to 6.0 reduced cell density, but had a minor affect on aldehyde production.     

Technological characteristics of yeast strains and their potential as starter adjuncts in Greek-style black olive fermentation

The technological properties of Debaryomyces hansenii (15 strains) and Torulaspora delbrueckii (32 strains) isolated from Greek-style black olives under conditions typical for black olive fermentation were studied. Furthermore, the killer character of the strains was assessed as well as their antimicrobial action against food-borne pathogens. All strains could grow at 15°C and low pH (2.5), whereas the majority of the strains were able to grow at 10% (w/v) NaCl, assimilated d-galacturonic acid, and showed lipolytic activity. Only 33% of D. hansenii and 9% of T. delbrueckii strains could hydrolyse 1% (w/v) oleuropein. A large majority of the strains tolerated 0.3% (w/v) bile salts, which in correlation with acid resistance indicates probiotic potential. Cross-reactions between culture filtrates of D. hansenii and T. delbrueckii and 56 yeast strains isolated during spontaneous Greek-style black olive fermentation were conducted. Focusing on their lytic activity, 17 mycogenic strains were selected. Culture filtrates of the mycogenic strains inhibited strains of L. monocytogenes, B. cereus and S. typhimurium. The active substance was heat resistant (stable after heating at 100°C for 10 min) as well as stable over a pH range from 4.0 to 6.5. The possible inhibition of undesirable yeast contaminants and food-borne pathogens in situ on fermented olives as well as the probiotic potential of strains used as starter adjuncts would contribute to the improvement of quality of the fermented product.     

Whey-cheese production using freeze-dried kefir culture as a starter

AIMS: The aim of the present study was to evaluate the use of a freeze-dried kefir culture in the production of a novel type of whey-cheese similar to traditional Greek Myzithra-cheese, to achieve improvement of the quality characteristics of the final product and the extension of shelf-life. METHODS AND RESULTS: The use of kefir culture as a starter led to increased lactic acid concentrations and decreased pH values in the final product compared with whey-cheese without starter culture. The effect of the starter culture on production of aroma-related compounds responsible for cheese flavour was also studied using the solid phase microextraction gas chromatography/mass spectrometry technique. Spoilage in unsalted kefir-whey-cheese was observed on the thirteenth and the twentieth day of preservation at 10 and 5 degrees C, respectively, while the corresponding times for unsalted whey-cheese preservation were 11 and 14 days. CONCLUSIONS: The cheeses produced were characterized as high-quality products during the preliminary sensory evaluation. An indication of increased preservation time was attributed to the freeze-dried kefir culture, which also seemed to suppress growth of pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggested the use of kefir culture as a means to extend the shelf-life of dairy products with reduced or no salt content.     

Development and evaluation of a starter culture for the industrial production of gari

Gari starter cultures (Gastat) were developed by mixing pure single strains of the organisms that ferment cassava. They were propagated and maintained as granules on dried cocoyam slurry. The cultures were tested for fermentative and acid-producing activity. The acidity produced at 30°C varied from 0.07% to 0.85% lactic acid with maximum levels occurring after 48 h. High levels of reducing sugar were produced during the first 24 h. The amounts produced were about 50% more than those from the self-inoculated cassava. The quality of the gari produced by the starter cultures was good and well accepted. The texture was similar to that produced by natural fermentation. These results highlight the possibility of using starter cultures in the large-scale production of gari.     

Determination of microbial diversity in Daqu, a fermentation starter culture of Maotai liquor, using nested PCR-denaturing gradient gel electrophoresis

This study endeavored to investigate the diversity of microbes present during the shaping, ripening and drying of Daqu, a fermentation starter culture and substrata complex of Maotai alcoholic spirit. A nested PCR-denaturing gradient gel electrophoresis technique was utilized with different combinations of primers. The results showed the presence of bacteria, yeasts and molds. The microflora, which originate from wheat, were readily detectable during every stage of the fermentation process. However, the microbial structure had clear differences in the shaping, ripening and drying processes. In the shaping stage, there was a high level of diversity of the LAB (lactic acid bacteria) and fungi in the shaped samples. In the ripening stage, however, a reduction of diversity of fungi with a high level of diversity of the Bacilli was observed in the ripened samples. In the drying stage, the diversity of Bacilli and fungi, especially acid-producing bacteria, reduced dramatically. Interestingly, uncultured Lactococcus sp., Microbacterium testaceum, Cochliobolus sp., and Thermoascus crustaceus were the first to be detected in the fermentation starters used in liquor production. This study revealed the microbial diversity and distributions during the shaping, ripening and drying of Daqu-making, facilitating evaluation of the hygienic conditions and aiding in the design of specific starter and/or adjunct cultures.