Elimination of Lactobacillus plantarum, Corynebacterium spp., Staphylococcus saprophyticus and Pseudomonas paucimobillis from micropropagated Hemerocallis, Choisya and Delphinium cultures using antibiotics

C. LEIFERT, H. CAMOTTA, S.M. WRIGHT, B. WAITES, V.A. CHEYNE & W.M. WAITES. 1991. The antibiotic sensitivity patterns of bacterial contaminants isolated from micropropagated plant cultures were determined. Lactobacillus plantarum, Corynebacterium spp., Staphylococcus saprophyticus and Pseudomonas paucimobilis were eliminated from plant tissue cultures of Hemerocallis, Choisya and Delphinium by incorporating combinations of antibiotics into the plant growth medium. Elimination of contaminants resulted in increased growth rates of all plant cultures, except Choisya infected with Staph. saprophyticus. Pseudomonas maltophilia was not eliminated from Delphinium by any of the antibiotic treatments tested.     

Lactobacillus plantarum; a deleterious contaminant of plant tissue cultures

Lactobacillus plantarum was isolated from in vitro plant cultures of Hemerocallis Stella d'ora, Catherine Woodbury and Stafford. Infected cultures deteriorated rapidly during three subcultures (15 weeks) when grown in vitro , showing chlorotic white shoots and grey calli. Weaned plants developed normally when transferred to the soil. The drop in multiplication rate of plant cultures coincided with a decrease in pH of the growth media. Uninfected plants of Hemerocallis Stella d'ora showed the same symptoms after inoculation with L. plantarum. Lactobacillus plantarum was reisolated from inoculated plant cultures that showed symptoms, thus fulfilling Koch's postulates. In plants inoculated with L. plantarum both the amount of dl-lactic acid formed and the number of plants showing symptoms increased with increasing numbers of bacteria in the inoculum, wheras plant multiplication rate and pH decreased. These effects could be reproduced by adding dl-lactic acid to the multiplication medium of plants free from L. plantarum , suggesting that the bacterial production of lactic acid was responsible for the changes in infected plants.     

Plasmid profiles of Lactobacillus plantarum spp. found as contaminants in Hemerocallis plant tissue cultures

Plasmid profiles of 30 strains of Lactobacillus plantarum isolated from 3-year-old tissue cultures and surface sterilized stem sections of glasshouse grown plants of three different varieties of Hemerocallis were compared to pinpoint the source of Lact. plantarum contamination in plant tissue cultures. Since the profiles of strains isolated from in vitro and in vivo plant material were generally identical, it is thought that the source of Lact. plantarum contamination is the plant material used to initiate plant tissue cultures.     

Arabinose fermentation by Lactobacillus plantarum in sourdough with added pentosans and alphaalpha-L-arabinofuranosidase: a tool to increase the production of acetic acid

Sixty-five strains of obligately and facultatively heterofermentative sourdough lactic acid bacteria were screened for their capacity to grow optimally in the presence of arabinose, ribose and xylose as carbon sources. Lactobacillus alimentarius 15F, Lact. brevis 10A, Lact. fermentum 1F and Lact. plantarum 20B showed higher growth rate, cell yield, acidification rate and production of acetic acid when some pentoses instead of maltose were added to the SDB medium. Lactobacillus plantarum 20B used arabinose also in a synthetic medium where complex growth factors such as yeast extract were omitted. Other Lact. plantarum strains did not show the same property. Pentosan extract was treated with alpha-L-arabinofuranosidase from Aspergillus niger or endo-xylanase from Bacillus subtilis to produce hydrolysates containing mainly arabinose and xylose, respectively. In particular, the hydrolysate containing arabinose substantiated the growth and the production of lactic acid and, especially, of acetic acid by Lact. plantarum 20B. Sourdough fermentation by Lact. plantarum 20B with addition of pentosan extract and alpha-L-arabinofuranosidase increased the acidification rate, titratable acidity and acetic acid content compared with traditional sourdough. A facultatively heterofermentative strain, Lact. plantarum 20B, also produced a sourdough with an optimal fermentation quotient.     

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.     

Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755

Phytase enzymes can increase the nutritional value of food and feed by liberating inorganic phosphate from phytate, the major storage form of phosphorus in plants. The phytase (phyC) from Bacillus subtilis VTT E-68013 was expressed in Lactobacillus plantarum strain 755 using Lact. amylovorus alpha-amylase secretion signals. In an overnight cultivation in MRS medium containing cellobiose for induction of the alpha-amylase promoter, catalytically active phytase was secreted as a predominant extracellular protein. However, Western blot analysis revealed unprocessed and processed phytase in the cell fraction. Pulse chase experiments showed that the recombinant phytase was secreted at a slower rate in comparison to the native proteins of Lact. plantarum 755.     

Microbiological events during commercial meat fermentations.

Microbiological developments during industrial meat fermentations (salami), made with and without commercial starter cultures, were followed at two factories in Germany and Italy. In the German product microbial growth was evident only for the first 48 h, followed by a gradual decline in numbers of most micro-organisms. The pH fell from 5.8 to 4.8 in the 28 d required for production. In Italy a similar situation was seen, except that a second period of bacterial growth began around 15 d, coincident with the appearance of intentional surface mould growth which reversed the pH fall, the final pH being 6.2. The German starter culture was a mixture of Lactobacillus plantarum and Staphylococcus carnosus, whereas in Italy only Staph. carnosus was used. The strain of Lact. plantarum used did not grow in the German product whereas the Staph. carnosus grew well in both products to form a substantial proportion of the final microflora.     

Microbiological events during commercial meat fermentations

Microbiological developments during industrial meat fermentations (salami), made with and without commercial starter cultures, were followed at two factories in Germany and Italy. In the German product microbial growth was evident only for the first 48 h, followed by a gradual decline in numbers of most micro-organisms. The pH fell from 5.8 to 4.8 in the 28 d required for production. In Italy a similar situation was seen, except that a second period of bacterial growth began around 15 d, coincident with the appearance of intentional surface mould growth which reversed the pH fall, the final pH being 6.2. The German starter culture was a mixture of Lactobacillus plantarum and Staphylococcus carnosus , whereas in Italy only Staph. carnosus was used. The strain of Lact. plantarum used did not grow in the German product whereas the Staph. carnosus grew well in both products to form a substantial proportion of the final microflora.     

Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum lp 31 strain isolated from dry-fermented sausage

Aims:  Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage.
Methods and Results:  Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5·0 to 6·0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558·85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus , Bacillus cereus and Listeria monocytogenes.
Conclusions:  The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition.
Significance and Impact of the Study:  In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.     

Selected nondigestible carbohydrates and prebiotics support the growth of probiotic fish bacteria mono-cultures in vitro

Aims:  To search for nondigestible but fermentable (NDF) carbohydrates and prebiotics with a potency to promote the growth of selected bacteria in vitro .
Methods and Results:  The growth of three reference bacteria strains Bacillus subtilis LMG 7135^T, Carnobacterium piscicola LMG 9839, Lactobacillus plantarum LMG 9211 and one candidate probiotic bacteria Lactobacillus delbrueckii subsp. lactis was investigated over a minimum period of 48 h in the presence of β -glucan, xylo-oligosaccharide, arabinoxylo-oligosaccharide, inulin, oligofructose and glucose. Besides the capability to grow on inulin and oligofructose containing media, a distinct high growth in β -glucan based substrates and a low growth in (arabino)xylooligosaccharide containing media were evident for most bacteria tested. With the exception of B. subtilis and L. plantarum , other bacteria grew equally well or even better on different substrates than on glucose. The fermentation of studied carbohydrates by these micro-organisms was dominated by the production of acetic acid as the main short chain fatty acid.
Conclusions:  Selected bacteria are able to ferment and grow on NDF and prebiotic carbohydrates but in a substrate dependent manner.
Significance and Impact of the Study:  This study delivers a first screening of which NDF or prebiotic carbohydrates are the most promising for aquaculture feed supplementations.     

A new strategy to apply Bacillus subtilis MA139 for the production of solid-state fermentation feed

Aim:  The study investigated the potential of using Bacillus subtilis MA139 in combination with Lactobacillus fermentum and Saccharomyces cerevisae to produce solid-state fermentation feed.
Methods and Results:  In a pure fermentation, B. subtilis MA139 was able to grow and synthesize antimicrobial substances at temperatures from 25 to 37°C and at a pH from 5·0 to 9·0. Subsequently, B. subtilis MA139, Lact. fermentum and S. cerevisae were used as starter strains co-inoculated in unsterilized substrate (feed-grade soybean meal and wheat bran). Following 10 days of fermentation in a newly developed plastic bag equipped with a one-way valve, lactic acid bacteria and Bacillus became the predominant strains while S. cerevisae cells decreased slightly. Enterobacteriaceae ( Escherichia coli K88 and Salmonella typhimurium ) were not detected.
Conclusions:  Use of B. subtilis MA139 as a starter strain co-inoculated with S. cerevisae and Lact. fermentum successfully controlled the growth of enterobacteriaceae.
Significance and Impact of the Study:  This study provided a facile and low-cost way to produce solid-state fermentation feed.     

Effect of medium acidity on growth and rooting of different plant species growing in vitro

Micropropagated Choisya, Daphne, Delphinium, Hemerocallis, Hosta, Iris and Photinia were found to adjust the pH of Murashige and Skoog's plant tissue culture medium (initial pH 5.6 or 3.5) to different values depending on the species. When plant growth and rooting rates were determined after plants had been grown on media initially adjusted or buffered to values between 2.6 and 5.7 the different plant species were also found to have distinct pH requirements for optimal growth and/or rooting rates.Abbreviations MS Murashige & Skoog's (1962) medium - MS19 MS with additionally 10 g l^–1 sucrose - 80 mg l^–1 adenine sulphate and 130.9 mg l^–1 NaH₂PO₄ - BA 6-benzyladenine - NAA 1-naphthyl-acetic acid - IBA 3-indole-butyric acid - IAA 3-indole-acetic acid - 2iP N₆(2-isopentyl) adenine     

Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates

AIMS: The overall growth kinetics of four potentially probiotic strains (Lactobacillus fermentum, Lact. reuteri, Lact. acidophilus and Lact. plantarum) cultured in malt, barley and wheat media were investigated. The objectives were to identify the main factors influencing the growth and metabolic activity of each strain in association with the cereal substrate. METHODS AND RESULTS: All fermentations were performed without pH control. A logistic-type equation, which included a growth inhibition term, was used to describe the experimental data. In the malt medium, all strains attained high maximum cell populations (8.10-10.11 log10 cfu ml(-1), depending on the strain), probably due to the availability of maltose, sucrose, glucose, fructose (approx. 15 g l(-1) total fermentable sugars) and free amino nitrogen (approx. 80 mg l(-1)). The consumption of sugars during the exponential phase (10-12 h) resulted in the accumulation of lactic acid (1.06-1.99 g l(-1)) and acetic acid (0.29-0.59 g l(-1)), which progressively decreased the pH of the medium. Each strain demonstrated a specific preference for one or more sugars. Since small amounts of sugars were consumed by the end of the exponential phase (17-43%), the decisive growth-limiting factor was probably the pH, which at that time ranged between 3.40 and 3.77 for all of the strains. Analysis of the metabolic products confirmed the heterofermentative or homofermentative nature of the strains used, except in the case of Lact. acidophilus which demonstrated a shift towards the heterofermentative pathway. All strains produced acetic acid during the exponential phase, which could be attributed to the presence of oxygen. Lactobacillus plantarum, Lact. reuteri and Lact. fermentum continued to consume the remaining sugars and accumulate metabolic products in the medium, probably due to energy requirements for cell viability, while Lact. acidophilus entered directly into the decline phase. In the barley and wheat media all strains, especially Lact. acidophilus and Lact. reuteri, attained lower maximum cell populations (7.20-9.43 log10 cfu ml(-1)) than in the malt medium. This could be attributed to the low sugar content (3-4 g l(-1) total fermentable sugar for each medium) and the low free amino nitrogen concentration (15.3-26.6 mg l(-1)). In all fermentations, the microbial growth ceased at pH values (3.73-4.88, depending on the strain) lower than those observed for malt fermentations, which suggests that substrate deficiency in sugars and free amino nitrogen contributed to growth limitation. CONCLUSIONS: The malt medium supported the growth of all strains more than barley and wheat media due to its chemical composition, while Lact. plantarum and Lact. fermentum appeared to be less fastidious and more resistant to acidic conditions than Lact. acidophilus and Lact. reuteri. SIGNIFICANCE AND IMPACT OF THE STUDY: Cereals are suitable substrates for the growth of potentially probiotic lactic acid bacteria.     

Malt sprout extract medium for cultivation of Lactobacillus plantarum protective cultures

AIMS: The aim was to develop a cheap cereal-based alternative medium for the large-scale production of biopreservative Lactobacillus plantarum VTT E-79098. We examined the effect of growth medium and pH control on the cell yield of Lact. plantarum E-79098 and the antimicrobial activity of the cell-free extracts. METHODS: Fermentations using a novel Malt Sprout Extract Medium (MSE) were performed with different pH regimes. The antimicrobial activity of the cell-free extracts against Pantoea agglomerans VTT E-90396 and Fusarium avenaceum VTT D-80147 was assessed with automated turbidometry. SIGNIFICANCE AND IMPACT OF THE STUDY: When compared with MRS, the MSE medium cultures produced equal growth yields of Lact. plantarum VTT E-79098 and enhanced antimicrobial potential against the Gram-negative bacterium P. agglomerans and a Fusarium fungus. The MSE medium can be used as a low-cost alternative to MRS for producing high cell yields and good antimicrobial activity of Lact. plantarum.     

Effects of concentrated supernatants recovered from Lactobacillus plantarum on Escherichia coli growth and on the viability of a human promyelocytic cell line

Aims:  The ability of concentrated supernatants from Lactobacillus plantarum to produce a disruption of plasma membrane in eukaryotic and prokaryotic cells has been examined.
Methods and Results:  A strain of Lact. plantarum (tolerant to acid and bile salts and resistant to several antibiotics) was used. It inhibited the growth of pathogenic Escherichia coli and L. monocytogenes . Supernatants from Lact. plantarum were concentrated by centrifugation. Either E. coli or HL-60 cells (a human promyelocytic cell line) were treated in the presence of the concentrated supernatants. The effect of concentrated supernatants from Lact. plantarum on E. coli growth demonstrated a bacteriostatic activity and a loss of cell viability measured by sytox green staining. Concentrated supernatants were capable of disturbing plasma membrane in E. coli and of promoting a cytotoxic and lyctic action on HL-60 cells and on human erythrocytes, respectively.
Conclusions:  These results suggest that Lact. plantarum release an effective compound responsible for an important effect in the disruption of E. coli plasma membrane and for a cytototoxic activity on promyelocytic leukaemia cells.
Significance and Impact of the Study:  This is the first in vitro study about the antimicrobial and biological activities of concentrated supernatants from Lact. plantarum .     

Growth of Lactobacillus plantarum in media containing hydrolysates of fish viscera

AIMS: To compare growth of Lactobacillus plantarum on media containing hydrolysates (peptones) from cod viscera with growth on commercial media. METHODS AND RESULTS: Growth of Lact. plantarum on various fish peptones and commercial peptones/extracts was evaluated using both a Bioscreen apparatus (microtiter plates, no pH control) and fermentors (with pH control). Generally, the performance of the fish peptones was good and only beaten by the performance of yeast extract. Replacement of the 22 g l(-1) complex nitrogen source in standard MRS medium with only 5 g l(-1) fish peptone reduced the biomass yield with only 10%, whereas replacement with a mixture of 2.5 g l(-1) fish peptone and 2.5 g l(-1) yeast extract increased the biomass yield by 10%. CONCLUSIONS: Peptones derived from cod viscera support excellent growth of Lact. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: We show that peptones derived from cod viscera are promising constituents of growth media for fastidious food bacteria such as lactobacilli. Media containing these peptones show excellent performance while problems associated with the use of meat-derived peptones (BSE, kosher status) or plant-derived peptones (genetically modified organisms) are avoided.     

Citrate metabolism by Lactobacillus plantarum isolated from orange juice

The behaviour of Strains of Lactobacillus plantarum isolated from fermented orange juice and Lact. plantarum DSM 20174 was studied in the presence of citrate. When used as sole carbon source, citrate scarcely supported the growth of the bacteria. It was shown to enhance the growth of Lact. plantarum in glucose media. Under acid conditions (pH 4·0–5·0), 1 mol of citrate yielded 1·7 mol of acetate as sole major final metabolite with release of CO₂ in the gas phase.     

The presence of prtP proteinase gene in natural isolate Lactobacillus plantarum BGSJ3–18

Aims:  The study of proteolytic activity and examination of proteinase gene region organization in proteolytically active Lactobacillus plantarum strains from different natural sources.
Methods and Results:  A set of 37 lactobacilli was distinguished by using multiplex PCR assay. Results showed that 34 strains were Lact. plantarum and three of them were Lact. paraplantarum . The examination of proteolytic activity revealed that 28 Lact.   plantarum and two Lact.   paraplantarum hydrolyse β-casein. Further analyses of all proteolytically active Lact. plantarum with primers specific for different types of CEPs demonstrated that strain BGSJ3–18 has prtP catalytic domain as well as prtP – prtM intergenic region showing more than 95% sequence identity with the same regions present in Lact. paracasei , Lact. casei and L. lactis . No presence of prtB , prtH or prtR proteinase genes was detected in any of tested Lact. plantarum strains.
Conclusions:  One out of 28 analysed Lact. plantarum strains harbours the prtP -like gene. The other proteolytically active Lact. plantarum probably possesses a different type of extracellular proteinase(s).
Significance and Impact of the Study:  It is the first report about the presence of the prtP –like gene in Lact. plantarum , which illustrates the mobility of this gene and its presence in different species.     

Growth of Bacillus cereus in fermenting tempeh made from various beans and its inhibition by Lactobacillus plantarum

Counts of Bacillus cereus reached ca 10(8) cfu/g within 40 h in fermenting unacidified horsebean tempeh and resulted in complete spoilage of the product. In fermenting unacidified pea, chickpea and soybean tempeh, B. cereus counts reached 10(6)-10(7) cfu/g, although the products were not spoiled. Inoculation of these unacidified beans with Lactobacillus plantarum decreased the final count of B. cereus by 2 log units, but had no effect on its growth in unacidified horsebean tempeh and its subsequent spoilage. Acidification of the beans during soaking resulted in a lower rate of B. cereus growth during fermentation. Inoculation of acidified beans with Lact. plantarum resulted in a markedly lower growth rate of B. cereus. In an associative broth culture study, B. cereus was completely inhibited by Lact. plantarum at pH values of about 5.5. Lactobacillus plantarum may be used to control the growth of B. cereus during tempeh production.     

Mechanisms of inhibition of Erwinia amylovora by Erw. herbicola in vitro and in vivo

The mechanisms by which Erwinia herbicola inhibits Erwinia amylovora , the fire blight pathogen, were investigated. The optimum pH for growth of Erw. amylovora strain Ea273 in nutrient-yeast extract-glucose broth (NYGB) was 7.0 and growth was markedly reduced at pH values below 6.0. In contrast, the growth rates of Erw. herbicola strains Eh141 and Eh112Y were only slightly reduced at pH levels as low as 4.5, relative to pH 6-8. When Ea273 was grown in NYGB in the presence of Eh141 or Eh112Y, the media became acidic and lower populations of Ea273 were recovered, compared with populations from buffered NYGB. Acidification of plant tissue as a consequence of growth of Erw. herbicola did not occur, however, and thus acid-based inhibition of growth in planta is unlikely. The growth rates of nine strains of Erw. herbicola and their abilities to reduce the pH of NYGB did not correlate with their different abilities to prevent development of fire blight incited by Ea273 in a research apple orchard. When grown in mixed culture, Eh114 and Eh112Y grew to higher populations than Ea273 due to depletion of a nitrogen source needed by Ea273. The ability of 12 strains of Erw. herbicola to produce antibiotics inhibitory to Ea273 on a glucose-asparagine medium correlated with the effectiveness of the strains in suppressing fire blight. A crude preparation of the Eh318 antibiotic delayed development of disease in immature pear fruits incited by Ea273 but not by strain Ea273R318, which is resistant in vitro to the Eh318 antibiotic. Cells of Eh318 protected immature pear fruits more effectively from infection by Ea273 than from the resistant strain Ea273R318.