Experiences with the use of a starter culture in the fermentation of maize for ‘kenkey’ production in Ghana

Controlled fermentation of maize was carried out using six strains of Lactobacillus fermentum and one strain of yeast, Saccharomyces cerevisiae, isolated from traditionally fermented maize dough as starter cultures for inoculum enrichement. The fermentations were monitored by pH, acidity, microbiological analysis and taste panel evaluation of two products, kenkey and koko, prepared from the fermented doughs. The strains of L. fermentum used as starter culture dominated the microflora during fermentation and in most inoculated doughs the required pH was attained by 24 h instead of 48 h of dough fermentation. Higher contents of lactic acid bacteria and yeasts were observed in inoculated doughs at the initial stages of fermentation but the spontaneously fermented doughs attained similar lactic acid bacteria and yeasts counts by 24 h of dough fermentation. The organoleptic quality of kenkey and koko prepared from doughs fermented with starter culture for 48 h was not significantly different from the traditional products. Kenkey prepared from doughs fermented for 24 h with starter culture were found to be unacceptable by the taste panel although similarly produced koko was acceptable.The authors are with the Food Research Institute, Council for Scientific and Industrial Research, P.O Box M 20. Accra, Ghana.     

A polyphasic study on the taxonomic position of industrial sour dough yeasts

The sour dough bread making process is extensively used to produce wholesome palatable rye bread. The process is traditionally done using a back-slopping procedure. Traditional sour doughs in Finland comprise of lactic acid bacteria and yeasts. The yeasts present in these doughs have been enriched in the doughs due to their metabolic activities, e.g. acid tolerance. We characterized the yeasts in five major sour bread bakeries in Finland. We found that most of the commercial sour doughs contained yeasts which were similar to Candida milleri on the basis of 18S rDNA and EF-3 PCR-RFLP patterns and metabolic activities. Some of the bakery yeasts exhibited extensive karyotype polymorphism. The minimum growth temperature was 8 degrees C for C. milleri and also for most of sour dough yeasts.     

The antimicrobial effect of organic acids, sour dough and nisin against Bacillus subtilis and B. licheniformis isolated from wheat bread

Prevention of growth in wheat bread for more than 6 d of approximately 10⁶ rope-producing Bacillus subtilis spores per gram of dough was achieved by addition of propionic or acetic acids at levels of 0·10% v/w (based on flour weight), or by addition of 15% sour dough fermented with Lactobacillus plantarum C11, Lact. brevis L62, Lact. plantarum ('vege-start 60'), Lact. plantarum (ch 20), Lact. maltaromicus (ch 15), or the commercial sour dough starter culture, Lact. sanfrancisco L99. These cultures resulted in an amount of total titratable acids above 10 in the sour dough and a pH value below 4·8 in the final bread. Bacteriocin-producing lactic acid bacteria added as starter cultures in wheat dough and nisin (Nisaplin) at levels up to 100 p.p.m. g⁻¹ flour had no effect against B. subtilis and B. licheniformis strains, despite the fact that nisin-producing strains of Lactococcus lactis ssp. lactis among 186 strains of lactic acid bacteria had demonstrated inhibitory activity against B. subtilis and B. licheniformis in an agar spot assay.     

Technological and molecular diversity of Lactobacillus plantarum strains isolated from naturally fermented sourdoughs

Thirty Lactobacillus (L.) plantarum strains, isolated from sourdough, were identified by biochemical tests as well as 16S rDNA sequencing and differentiated on the basis of technological properties, such as amylase, protease, phytase and antirope activities. These properties were shown to be widely differing among the strains, indicating a significant technological diversity. Genetic differentiation was achieved by restriction endonuclease analysis-pulsed field gel electrophoresis (REA-PFGE) that allowed the L. plantarum strains to be divided into 10 different genomic groups. Moreover, 32 different starters were employed in dough making experiments; each starter consisted of a single strain of L. plantarum associated with a maltose positive or a maltose negative yeast. The technological properties of the doughs were greatly influenced by the type of strain included in the starter. The time of leavening and the acidification activities detected in the dough were enhanced by the presence of L. plantarum strains. The bacterial and yeast contents and fermentation properties were statistically treated by principal component analysis (PCA), which allowed the discrimination of different typologies of dough. The study of the peculiar characteristics of different strains of L. plantarum is fundamental for a better understanding of their potential in affecting the nutritional value, quality and stability of the baked goods. L. plantarum strains are able to differentially influence the dough quality when employed as starters.     

Superior molasses assimilation, stress tolerance, and trehalose accumulation of baker's yeast isolated from dried sweet potatoes (hoshi-imo)

Yeast strains were isolated from dried sweet potatoes (hoshi-imo), a traditional preserved food in Japan. Dough fermentation ability, freeze tolerance, and growth rates in molasses, which are important characteristics of commercial baker's yeast, were compared between these yeast strains and a commercial yeast derivative that had typical characteristics of commercial strains. Classification tests including pulse-field gel electrophoresis and fermentation/assimilation ability of sugars showed that almost the stains isolated belonged to Saccharomyces cerevisiae. One strain, ONY1, accumulated intracellular trehalose at a higher level than commercial strain T128. Correlated with intracellular trehalose contents, the fermentation ability of high-sugar dough containing ONY1 was higher. ONY1 also showed higher freeze tolerance in both low-sugar and high-sugar doughs. The growth rate of ONY1 was significantly higher under batch and fed-batch cultivation conditions using either molasses or synthetic medium than that of strain T128. These results suggest that ONY1 has potential commercial use as baker's yeast for frozen dough and high-sugar dough.     

Microorganisms of the San Francisco Sour Dough Bread Process: I. Yeasts Responsible for the Leavening Action

Two hundred isolates from San Francisco sour dough French bread fermentations (40 from each of five different bakeries) were screened by fermentation tests and for their ability to grow in the presence of cycloheximide (Actidione). All of the isolates from four of the bakeries and 70% of those from the fifth were unable to utilize maltose but grew well on other sugars, even in the presence of cycloheximide. The remaining few isolates from the fifth bakery utilized maltose but not galactose and were inhibited by cycloheximide. No bakers' yeast types were found. Sixteen of the maltose-negative and five of the galactose-negative isolates were subjected to more rigorous taxonomic procedures. All of the maltose-negative isolates were identified as asporogenous strains of Saccharomyces exiguus (Torulopsis holmii) and the galactose-negative ones, as S. inusitatus. The predominance of S. exiguus, its vigor in the particular acidic environment of the sour dough, and the correlation of its numbers with the leavening function constitute strong evidence on the role of this organism in the sour dough system.     

Occurrence of megaplasmids in halobacteria

Sixty-five halobacteria, including culture collection and freshly isolated strains from widely differing geographical areas, were examined for the presence of high molecular weight plasmids by agarose gel electrophoresis. Seventy-five per cent of all the strains were shown to harbour at least one plasmid. In the majority of strains three or four megaplasmids were detected. Approximate molecular weights of the plasmids were in the range < 100 to 300 megadaltons (Mdal). In most culture collection strains, two or three plasmids were demonstrated, except in two in which no plasmid was detected, and in two Haloarcula strains which were found to contain five and eight plasmids; four and six of the latter were more than 100 Mdal. No relationship between the plasmid profile of each strain and its taxonomic assignation nor its isolation source was found. Evidence is presented for the first time on the occurrence of megaplasmids in halobacteria.     

Isolation and characterization of new amylolytic strains of Lactobacillus fermentum from fermented maize doughs (mawè and ogi) from Benin

Twelve amylolytic heterofermentative lactic acid bacteria were isolated in Benin from the fermentation processes of maize sour dough, namely ogi and mawè. Discrimination of strains was performed by DNA restriction patterns and compared with carbohydrate fermentation profiles. This allowed two new amylolytic strains, Ogi E1 and Mw2, belonging to the species Lactobacillus fermentum , to be distinguished. Strains Ogi E1 and Mw2 presented different amylolytic activities; amylase from strain Mw2 was more acidophilic and mesophilic than amylase produced by strain Ogi E1.     

Stress tolerance in doughs of Saccharomyces cerevisiae trehalase mutants derived from commercial Baker's yeast.

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Deltanth1), acid trehalase mutants (Deltaath1), and double mutants (Deltanth1 ath1) by using commercial baker's yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Deltanth1 and Deltaath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Deltanth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.     

Antifungal activity of Leuconostoc citreum and Weissella confusa in rice cakes

The antifungal activity of organic acids greatly improves the shelf life of bread and bakery products. However, little is known about the effect of lactic acid fermentation on fungal contamination in rice cakes. Here, we show that lactic acid fermentation in rice dough can greatly retard the growth of three fungal species when present in rice cakes, namely Cladosporium sp. YS1, Neurospora sp. YS3, and Penicillium crustosum YS2. The antifungal activity of the lactic acid bacteria against these fungi was much better than that of 0.3% calcium propionate. We found that organic acids including lactic and acetic acid, which are byproducts of lactic fermentation or can be artificially added, were the main antifungal substances. We also found that some Leuconostoc citreum and Weissella confusa strains could be good starter species for rice dough fermentation. These results imply that these lactic acid bacteria can be applicable to improve the preservation of rice cakes.     

Plasmid profiles and randomly amplified polymorphic DNA analysis of Salmonella enterica serotype Enteritidis strains from outbreaks and sporadic cases in Turkey

The aim of the study was to investigate the characteristics of Salmonella serotype Enteritidis strains isolated from outbreaks and sporadic cases in Turkey by plasmid profiles and randomly amplified polymorphic DNA (RAPD) patterns. A total of 64 S. Enteritidis clinical strains were selected from the culture collection of the Enterobacteria Laboratory of Ankara University Medical School Department of Microbiology and Clinical Microbiology for molecular analysis using the plasmid profiles and RAPD method. Fifty-six isolates (88%) harbored one to four plasmids ranging in size from 2.5 to 100 kbp. 57 kbp plasmids were the most common plasmids, and forty-four strains (69%) carried 57 kbp plasmids alone or together with other plasmids. The outbreak strains carried the same plasmid profile: three plasmids sized 57, 40, 3.0 kbp. None of the strains analyzed displayed any RAPD bands with the primer OPB-17. By using primer p-1254, 42 strains (66%) were divided into fourteen RAPD patterns. Ten of the outbreak strains (77%) showed >80% similarity by cluster analysis program. Analysis of RAPD-PCR with primer p-1254 proved an easy, rapid and discriminative method complementing antibiogram and plasmid profiles in routine laboratories, and may contribute to the investigations of S. Enteritidis which still cause outbreaks in Turkey. This study presents the first report on S. Enteritidis isolates in Turkey investigated by plasmid profiles and RAPD methods.     

Multiphasic approach to study the bacterial ecology of fermented sausages inoculated with a commercial starter culture

In this paper, the ability of a commercial starter culture to perform a sausage fermentation is evaluated. Molecular analysis revealed the presence of several strains of the same species contained in the starter culture with different behavior during the fermentation, and the contribution of Lactobacillus curvatus, which was only marginally isolated during the transformation.     

Principal-Component Analysis of the Characteristics Desirable in Baker's Yeasts

Twenty-seven properties considered to be required for good bakery products were examined in 56 industrial and 2 laboratory yeast strains. The data obtained were applied to principal-component analysis, one of the multivariate statistical analyses. The first and second principal components together were extracted, and these accounted for 77.7% of the variance. The first principal component was interpreted as the glycolytic activity of yeast in dough, and the second one was interpreted as the balance of leavening abilities in sweet and flour doughs from the factor loadings. The scattergram on the two principal components was effective in grouping the 58 yeast strains used.     

Principal-Component Analysis of the Characteristics Desirable in Baker's Yeasts

Twenty-seven properties considered to be required for good bakery products were examined in 56 industrial and 2 laboratory yeast strains. The data obtained were applied to principal-component analysis, one of the multivariate statistical analyses. The first and second principal components together were extracted, and these accounted for 77.7% of the variance. The first principal component was interpreted as the glycolytic activity of yeast in dough, and the second one was interpreted as the balance of leavening abilities in sweet and flour doughs from the factor loadings. The scattergram on the two principal components was effective in grouping the 58 yeast strains used.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27.5 degrees C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10(4) spores/g whereas with 800 g loaves survival occurred with doughs containing 5.0 X 10(3) spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0.2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

Multiphasic Approach To Study the Bacterial Ecology of Fermented Sausages Inoculated with a Commercial Starter Culture

In this paper, the ability of a commercial starter culture to perform a sausage fermentation is evaluated. Molecular analysis revealed the presence of several strains of the same species contained in the starter culture with different behavior during the fermentation, and the contribution of Lactobacillus curvatus, which was only marginally isolated during the transformation.     

Identification of lactose fermentation plasmids of streptococcal dairy starter strains by Southern hybridization

Twenty-two strains of Streptococcus cremoris , seven strains of Streptococcus lactis and three strains of Streptococcus lactis subsp. diacetilactis, each with a different plasmid complement, were isolated from a starter culture used in a Finnish dairy plant. By using DNA-DNA hybridization, with cloned 6-P-ß-galactosidase gene of the Strep, lactis plasmid pLP712 as a probe, the lactose fermentation genes were located, in each strain, in the large ( 30 MD) plasmid.     

Development and application of a multiple typing system for Clostridium difficile.

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

Plasmids in avirulent strains of Agrobacterium.

Twelve strains of Agrobacterium radiobacter isolated from naturally occurring crown galls or soil were found to be avirulent on sunflower, tomato, Kalanchoe, and carrot. Eleven strains contained plasmids of molecular weights 77 X 10(6) to 182 X 10(6) as determined by electron microscopy. One strain contained only a smaller plasmid (50 X 10(6) daltons). Several strains had both large and small (ca. 11 X 10(6) daltons) plasmids; one strain contained two large plasmids (112 X 10(6) and 136 X 10(6) daltons). Hybridization reactions of virulence plasmids from Agrobacterium tumefaciens strains C58 and A6 with plasmids from each of the A. radiobacter strains revealed that some A. radiobacter plasmids had less than 10% homology to either the C58 or A6 plasmids. Plasmids from some strains had approximately 50% homology with the C58 plasmid, but only one A. radiobacter plasmid contained more than 10% homology to the A6 plasmid. The presence of large plasmids in A. radiobacter strains did not correlate with sensitivity to agrocin 84; however, the utilization of the amino acid derivatives octopine and nopaline was generally correlated to partial base sequence homology to the C58 plasmid. We conclude that all large plasmids found in Agrobacterium strains are not virulence associated, although they may share base sequence homology with a virulence-associated plasmid. Further, plasmids from tumorigenic strains may be more closely related by base sequence homology to plasmids from nonpathogenic strains than to plasmids from other pathogenic strains.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27·5°C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10⁴ spores/g whereas with 800 g loaves survival occurred with doughs containing 5·0 times 10³ spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0·2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.