A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Avoparcin and vancomycin: useful antibiotics for the isolation of brewery lactic acid bacteria

Vancomycin (30 μg) and avoparcin (20 μg) have no effect on growth of hetero-fermentative lactobacilli, mesophilic homofermentative lactobacilli, Lactobacillus salivarius and bacteria of the genera Leuconostoc and Pediococcus. The growth of thermophilic homofermentative lactobacilli (with the exception of Lacto salivarius ), enteric and lactic streptococci and all other Gram-positive bacteria encountered in breweries (and likely to be encountered in foods) is inhibited by these antibiotics. The recovery of sub-lethally stressed cells is slightly reduced by the presence of either antibiotic. Vancomycin and avoparcin are useful selective agents for the detection of lactic acid bacteria in the brewing industry and may also be of use, for quality control purposes, in the distilling, wine, cider, dairy and meat industries. These antibiotics may also find applications in differentiation of pediococci from streptococci, and in rapid identification of lactobacilli.     

Effects of lactobacilli on yeast-catalyzed ethanol fermentations.

Normal-gravity (22 to 24 degrees Plato) wheat mashes were inoculated with five industrially important strains of lactobacilli at approximately 10(5), approximately 10(6), approximately 10(7), approximately 10(8), and approximately 10(9) CFU/ml in order to study the effects of the lactobacilli on yeast growth and ethanol productivity. Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus #3, Lactobacillus rhamnosus, and Lactobacillus fermentum were used. Controls with yeast cells but no bacterial inoculation and additional treatments with bacteria alone inoculated at approximately 10(7) CFU/ml of mash were included. Decreased ethanol yields were due to the diversion of carbohydrates for bacterial growth and the production of lactic acid. As higher numbers of the bacteria were produced (depending on the strain), 1 to 1.5% (wt/vol) lactic acid resulted in the case of homofermentative organisms. L. fermentum, a heterofermentative organism, produced only 0.5% (wt/vol) lactic acid. When L. plantarum, L. rhamnosus, and L. fermentum were inoculated at approximately 10(6) CFU/ml, an approximately 2% decrease in the final ethanol concentration was observed. Smaller initial numbers (only 10(5) CFU/ml) of L. paracasei or Lactobacillus #3 were sufficient to cause more than 2% decreases in the final ethanol concentrations measured compared to the control. Such effects after an inoculation of only 10(5) CFU/ml may have been due to the higher tolerance to ethanol of the latter two bacteria, to the more rapid adaptation (shorter lag phase) of these two industrial organisms to fermentation conditions, and/or to their more rapid growth and metabolism. When up to 10(9) CFU of bacteria/ml was present in mash, approximately 3.8 to 7.6% reductions in ethanol concentration occurred depending on the strain. Production of lactic acid and a suspected competition with yeast cells for essential growth factors in the fermenting medium were the major reasons for reductions in yeast growth and final ethanol yield when lactic acid bacteria were present.     

Selective control of lactobacilli in kimchi with nisin

The use of nisin to control the lactobacilli responsible for the over-ripening of kimchi, traditional Korean fermented vegetables, was studied. Of the 40 strains of lactic acid bacteria studied, most were sensitive to nisin at a concentration of 100 IU ml-1, while two strains appeared to be resistant. In MRS broth containing nisin at concentrations of 100-300 IU ml-1, the growth of sensitive strains of Lactobacillus plantarum was delayed for 2-3 d at 20 degrees C. When nisin was added to kimchi at a concentration of 100 IU ml-1, the growth of Lactobacillus spp. was inhibited more than the growth of Leuconostoc spp. Scanning electron micrograph observations confirmed the results, demonstrating the predominance of cocci in kimchi containing nisin. These results suggest that at recommended levels, nisin can be used to preserve kimchi by inhibiting lactobacilli more effectively than other lactic acid bacteria involved in kimchi fermentation.     

Citric acid metabolism in hetero- and homofermentative lactic acid bacteria.

The effect of citrate on production of diacetyl and acetoin by four strains each of heterofermentative and homofermentative lactic acid bacteria capable of utilizing citrate was studied. Acetoin was quantitatively the more important compound. The heterofermentative bacteria produced no acetoin or diacetyl in the absence of citrate, and two strains produced traces of acetoin in its presence. Citrate stimulated the growth rate of the heterofermentative lactobacilli. Acidification of all heterofermentative cultures with citric acid resulted in acetoin production. Destruction of accumulated acetoin appeared to coincide with the disappearance of citrate. All homofermentative bacteria produced more acetoin and diacetyl in the presence of citrate than in its absence. Citrate utilization was begun immediately by the streptococci but was delayed until at least the middle of the exponential phase in the case of the lactobacilli.     

Citric acid metabolism in hetero- and homofermentative lactic acid bacteria.

The effect of citrate on production of diacetyl and acetoin by four strains each of heterofermentative and homofermentative lactic acid bacteria capable of utilizing citrate was studied. Acetoin was quantitatively the more important compound. The heterofermentative bacteria produced no acetoin or diacetyl in the absence of citrate, and two strains produced traces of acetoin in its presence. Citrate stimulated the growth rate of the heterofermentative lactobacilli. Acidification of all heterofermentative cultures with citric acid resulted in acetoin production. Destruction of accumulated acetoin appeared to coincide with the disappearance of citrate. All homofermentative bacteria produced more acetoin and diacetyl in the presence of citrate than in its absence. Citrate utilization was begun immediately by the streptococci but was delayed until at least the middle of the exponential phase in the case of the lactobacilli.     

Investigations on the Glycerol Metabolism of Lactobacilli

S ummary . The glycerol metabolism of homofermentative and heterofermentative strains of Lactobacillus obtained either from Culture Collections or isolated from sausages has been investigated.
The results show that the homofermentative lactobacilli ( Thermobacterium and Streptobacterium ) produce mannose phosphate which is then metabolized to lactic acid and other byproducts.     

Enrichment of an alkaline-adapted association of streptococci and lactobacilli

By gradually increasing the starting pH during subsequent enrichment test runs, an association of lactic acid bacteria adapted readily to alkaline pH values. An association of homofermentative lactobacilli and streptococci capable of initiating growth at pH 10˙0 and rapidly producing lactic acid (3˙11 g/l/d) down to a final pH of ca 4˙0 was thus obtained. The streptococci present in the enriched association were capable of growing at pH 9˙5, whereas the lactobacilli were only capable of surviving at such high pH values.     

Effect of initial glucose concentration and inoculation level of lactic acid bacteria in shrimp waste ensilation

Fermentation conditions and microorganisms were determined, based on acid production, glucose concentration as carbohydrate source. Inoculation levels to obtain a stable shrimp waste silage were also determined. Shrimp waste ensilation was an efficient method of preservation, allowing the recovery of chitin and another added-value products such as pigments, proteins and enzymes. From the various lactic acid bacteria tested, Lactobacillus pentosus and Lactobacillus sp. (B2) were the best lactic acid producers, although small quantities of acetic acid were detected in samples inoculated with Lactobacillus pentosus. Therefore B2 was chosen for the analysis of glucose consumption as well as for the determination of optimum inoculation levels. The best results were obtained at 10% (w/w wet basis) and 5% (v/w wet basis) respectively. Presence of starters and initial glucose concentration were critical factors in the fermentation of shrimp waste. High initial glucose and starter concentrations reduced the time and increased the amount of lactic acid produced. The fermentation pattern changed during ensilation from hetero to homofermentative. Shrimp waste ensilation prevented the growth of spoilage microorganisms keeping their microbial counts steady and pH values within the acid region.     

Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria

Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.     

The aerobic growth and product formation of Lactobacillus,Leuconostoc, Brochothrix,and Carnobacterium in batch cultures

Summary The aerobic growth and metabolism of eleven homofermentative and three heterofermentative Lactobacillus strains, three Leuconostoc strains, two Brochothrix thermosphacta strains and two Carnobacterium strains were studied in batch cultures at pH 6.0 and 25°C on a complex substrate containing 10.0 g glucose per litre. All strains, except Carnobacterium divergens 69, grew well aerobically. An oxygen consumption was registered for 18 of the strains—the exceptions being Lactobacillus alimentarius DSM 20249^T, Lactobacillus farciminis DSM 20284^T and Lactobacillus sharpeae DSM 20505^T. The homofermentative lactobacilli showed a maximal oxygen consumption during the stationary growth phase and this was coupled with a low final viable count. Leuconostoc strains, heterofermentative lactobacilli, Brochothrix thermosphacta and Carnobacterium strains showed a maximal oxygen consumption during the exponential growth phase together with a high final viable count. The maximum specific growth rate varied from 0.19 to 0.54 h^-1 while the growth yield varied from 19 to 86 g dry weight per mol glucose consumed. In general, homofermentative lactobacilli produced dl-lactic acid, acetic acid and acetoin. The three heterofermentative lactobacilli produced dl-lactic acid and acetic acid, two strains also produced ethanol Leuconostoc spp. formed d-lactic acid, acetic acid, and ethanol. B. thermosphacta produced acetoin, acetic acid, formic acid, isobutyric acid and isovaleric acid but no lactic acid. Carnobacterium produced l-lactic acid, acetic acid and acetoin. All strains accumulated hydrogen peroxide except L. alimentarius DSM 20249^T, Carnobacterium piscicola 3 and B. thermosphacta.née Blickstad     

Induced lactic acid fermentation during the preservation stage of ripe olives from Hojiblanca cultivar

The influence of initial sodium chloride concentration (6 and 0%, w/v), acetic acid concentration (0.6, 0.3 and 0.0%, v/v), type of process (natural and inoculated), and storage system (anaerobic and aerobic) on the inducement of a lactic fermentation for the preservation stage of Hojiblanca cultivar ripe olives was investigated. The addition of 6% NaCl prevented colonization by lactic acid bacteria in all cases. A high level of acetic acid (0.6%) was effective in preserving olives for 2 months, although yeast growth was not inhibited for longer periods of storage. Natural growth of Lactobacillus plantarum did not occur. Inoculation with this micro-organism was effective only in the two treatments with tap water (with no NaCl) as the initial covering solution, although survival was reduced to a half of the added organisms when the initial pH was corrected with 0.3% acetic acid. In these two treatments pH quickly reached appropriate values (<4.0) for olive stabilization. Aerobic conditions led to low concentrations of carbon dioxide, without disturbing growth of lactic acid bacteria. Thus, the aerobic lactic acid fermentation, with tap water initially, was the most adequate preservation procedure for the storage of ripe olives prior to their oxidation treatment. Results of trials conducted on an industrial scale showed the same pattern and confirmed the viability of the new procedure.     

乳杆菌代谢产物对化脓性链球菌的抑制作用

【目的】分析乳杆菌代谢产物对化脓性链球菌的抑制作用。【方法】基于双层平板打孔法,通过测量抑菌圈大小来检测乳杆菌代谢产物对化脓性链球菌的抑菌作用;然后分别采用高效液相色谱法和4-氨酰安替比林法检测乳杆菌代谢产物中的有机酸和H2O2含量;最后,检测乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)、最小杀菌浓度(MBC)。【结果】对化脓性链球菌的抑菌效果以植物乳杆菌KLDS1.0667最好,副干酪乳杆菌KLDS1.0342-1次之,瑞士乳杆菌KLDS1.0203抑菌效果最差;乳酸和乙酸产量KLDS1.0667>KLDS1.0342-1>KLDS1.0203;H2O2产量KLDS1.0203>KLDS1.0667>KLDS1.0342-1。在抑菌试验中,乳杆菌的发酵上清液经去除H2O2处理后抑菌圈直径都减小;将发酵上清液的p H调至7.0后均检测不到抑菌圈。结果表明,乳杆菌代谢产物中对化脓性链球菌起抑制作用的主要物质为有机酸和H2O2,其中乳酸是产生抑菌作用的最主要物质。乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)分别为1.28、0.64和0.008 g/L,对化脓性链球菌的最小杀菌浓度(MBC)分别为5.12、2.56和0.032 g/L。【结论】乳杆菌可利用其代谢产物对化脓性链球菌产生抑制作用,主要抑菌物质为有机酸和H2O2。     

Batch and continuous production of lactic acid from salt whey using free and immobilized cultures of lactobacilli

Salt whey permeate was used as a substrate for lactic acid production by different strains of homofermentative lactobacilli. An isolate from Egyptian Cheddar cheese proved to be the most effective lactic acid producer. The salt whey permeate was optimized by addition of yeast extract and minerals to enable exponential growth of organisms. The lactic acid productivity of free and immobilized cells was compared and fermentation conditions were improved. Continuous lactic acid fermentation from salt whey permeate with cells immobilized in agarose beads was successfully carried out in a chemostat with a steady lactic acid concentration of 33.4 mg/ml.     

Evolution of the lactic acid bacterial community during malt whisky fermentation: a polyphasic study.

The development of the lactic acid bacterial community in a commercial malt whisky fermentation occurred in three broad phases. Initially, bacteria were inhibited by strong yeast growth. Fluorescence microscopy and environmental scanning electron microscopy revealed, in this early stage, both cocci and rods that were at least partly derived from the wort and yeast but also stemmed from the distillery plant. Denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA genes and sequence analysis revealed cocci related to Streptococcus thermophilus or Saccharococcus thermophilus, Lactobacillus brevis, and Lactobacillus fermentum. The middle phase began 35 to 40 h after yeast inoculation and was characterized by exponential growth of lactobacilli and residual yeast metabolism. Lactobacillus casei or Lactobacillus paracasei, L. fermentum, and Lactobacillus ferintoshensis were detected in samples of fermenting wort examined by DGGE during this stage. Bacterial growth was accompanied by the accumulation of acetic and lactic acids and the metabolism of residual maltooligosaccharides. By 70 h, two new PCR bands were detected on DGGE gels, and the associated bacteria were largely responsible for the final phase of the fermentation. The bacteria were phylogenetically related to Lactobacillus acidophilus and Lactobacillus delbrueckii, and strains similar to the former had previously been recovered from malt whisky fermentations in Japan. These were probably obligately homofermentative bacteria, required malt wort for growth, and could not be cultured on normal laboratory media, such as MRS. Their metabolism during the last 20 to 30 h of fermentation was associated with yeast death and autolysis and further accumulation of lactate but no additional acetate.     

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.     

Isolation of lactic acid bacteria from Malaysian foods and assessment of the isolates for industrial potential

Two traditional fermented food 'tapai' (fermented tapioca) and 'tempoyak' (fermented durian flesh), chilli puree and fresh goat's milk were used as sources for the isolation of lactic acid bacteria (LAB). A total of 126 isolates were obtained and by sequential screening for catalase activity and Gram-staining, 55 were determined to be LAB out of which 16 were established to be homofermentative by the gel plug test. Seven isolates were identified by use of the API 50CHL kit and two lactobacilli strains and one lactococci strain were selected to study their growth and lactic acid production profiles in a time course experiment. The lactobacilli strains, both isolated from 'tapai', produced higher amounts of cells and lactic acid from glucose as compared to the lactococci strain isolated from fresh goat's milk.     

Microflora of the aerobic preservation of directly brined green olives from Hojiblanca cultivar

New procedures for the preservation stage of ripe olives from Hojiblanca cultivar were studied. An aerobic fermentative process was used with initial pH correction (0.3% acetic acid) and various NaCl concentrations: 6, 3 and 0% (w/v) in tap water. Treatments were carried out at industrial level and the spontaneous changes monitored. At initial salt concentrations of 6 and 3% (w/v) NaCl, pH rose progressively, reaching 4.3 at equilibrium maintaining during this period a constant free lactic acidity of around 0.4% (w/v). When the initial solution was tap waste, however, the pH decreased rapidly to stabilize at about 3.7, and lactic acidity increased continuously to reach values over 1% (w/v) at the end of the preservation process. In all treatments aeration effectively purged the carbon dioxide from the preservation brines, preventing shrivelling of olives. The microbial growth was strongly influenced by the initial NaCl concentration. At 6 and 3%, only yeasts grew, the most abundant being Pichia membranaefaciens, P. vini, P. fermentans and Hansenula polymorpha. However, when there was no NaCl, lactic acid bacteria colonized the solution. Lactobacillus plantarum and Pediococcus inopinatus were the only species found. In this case there was a co-existence between yeasts and lactic acid bacteria. As the treatment that supported lactic acid bacteria achieved the best final pH and acidity for olive stability, it may help to overcome the obstacles to a lactic fermentative process during the preservation stage of ripe olives from the Hojiblanca cultivar.     

Lactic fermentation during the storage of 'Alorena' cultivar untreated green table olives

The development of lactic fermentation processes for the storage of directly brined olives (Aloreña cultivar) was investigated by three procedures: (1) a modification of the traditional method with an initial brine containing 9% (w/v) NaCl and 0.2% (w/v) acetic acid; (2) induced lactic fermentation with 6% NaCl and 0.2% acetic acid; and (3) conservation in acidified brine containing 6% NaCl and 0.6% acetic acid. In all cases, strains of Lactobacillus plantarum and Pediococcus spp. were present in each, indicating the great tolerance of these micro-organisms to high levels of lactic and acetic acids. They also appeared in an altered sequence. Counts of Pediococcus remained moderate (higher than Lact. plantarum ) throughout the last part of the preservation period. A commercial starter improved colonization by Lact. plantarum. Yeasts coexisted with the lactic bacteria throughout the preservation period although their importance in the fermentation process was very limited. The brine characteristics obtained after fermentation were suitable for assured product preservation. There was no spoilage. These results encourage research on the mechanism of lactic acid bacteria inhibition in brines and the development of lactic fermentation processes for directly brined olives from other olive cultivars.     

The effect of chlorogenic, gallic and quinic acids on the growth of spoilage strains of Lactobacillus collinoides and Lactobacillus brevis

The naturally occurring complex organic acids, chlorogenic acid, gallic acid and quinic acid, at concentrations of 100, 500 and 1000 mg l^-1 were evaluated for effects on the growth of three spoilage strains of Lactobacillus collinoides and one of Lact. brevis in acid tomato broth containing 5% (v/v) ethanol at pH 4.8. During early stages of growth, all the complex acids at each concentration stimulated growth of Lact. collinoides but not of Lact. brevis. During stationary phase, chlorogenic and gallic acids produced greater cell densities of all strains, whereas quinic acid generally had less effect. The presence of these complex acids in fruit products may increase the requirement for added preservative in order to prevent spoilage by certain strains of lactic acid bacteria.