Heterologous production of bacteriocins by lactic acid bacteria

Over the last two decades, bacteriocins produced by lactic acid bacteria (LAB) have been the subject of considerable research and industrial interest due to their potential as food biopreservatives. The development of heterologous expression systems for such antimicrobial compounds may offer a number of advantages over native systems, such as facilitating the control of bacteriocin gene expression or achieving higher production levels. In addition, the heterologous production by food-grade LAB offers an attractive method for overcoming some of the adverse situations that may affect the effectiveness of some bacteriocins in food systems. Construction of multibacteriocinogenic strains or acquisition of antimicrobial properties by industrial strains are further objectives that can be achieved through the use of heterologous gene expression systems. The development of new biotechnological tools and recent advances in LAB genetics account for the escalating number of studies dealing with heterologous production of bacteriocins by such hosts. This paper reviews the literature published on the subject and compares the different experimental strategies that have been used up to the present for this purpose.     

Factors influencing the production of volatile phenols by wine lactic acid bacteria

This work aimed to evaluate the effect of certain factors on the production of volatile phenols from the metabolism of p-coumaric acid by lactic acid bacteria (LAB) (Lactobacillus plantarum, L. collinoides and Pediococcus pentosaceus). The studied factors were: pH, L-malic acid concentration, glucose and fructose concentrations and aerobic/anaerobic conditions. It was found that, in the pH range of 3.5 to 4.5, the higher the pH the greater the production of volatile phenols. This behaviour is correlated with the effect of pH on bacterial growth. Increasing levels of L-malic acid in the medium diminished the production of 4-vinylphenol (4VP) and stimulated the production of 4-ethylphenol (4EP) by L. plantarum NCFB 1752 and L. collinoides ESB 99. The conversion of 4VP into 4EP by the activity of the vinylphenol reductase may be advantageous to the cells in the presence of L-malic acid, presumably due to the generation of NAD+, a cofactor required by the malolactic enzyme. Relatively high levels of glucose (20 g/L) led to an almost exclusive production of 4VP by L. plantarum NCFB 1752, while at low concentrations (≤ 5 g/L), 4EP is mainly or solely produced. Part of the glucose may be diverted to the production of mannitol as an alternative pathway to regenerate NAD+. This is corroborated by the experiments done with fructose, a compound that can be used as an electron acceptor by some bacteria becoming reduced to mannitol. In anaerobiosis, the reduction of 4VP into 4EP is clearly favoured, which is consistent with the need to increase the availability of NAD+ in these conditions. This study shows that the amount and the ratio 4VP/4EP produced by LAB are greatly affected by certain environmental and medium composition factors. The behaviour of the bacteria seems to be driven by the intracellular NAD+/NADH balance.     

Antagonism of Helicobacter pylori by bacteriocins of lactic acid bacteria

Antimicrobial activity of seven bacteriocins produced by lactic acid bacteria against Helicobacter pylori strains (ATCC 43504, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [DSM] 4867, DSM 9691, and DSM 10242) was investigated in vitro using a broth microdilution assay. The bacteriocins chosen for the study were nisin A; lacticins A164, BH5, JW3, and NK24; pediocin PO2; and leucocin K. Antimicrobial activity of the bacteriocins varied among the H. pylori strains tested, of which strain ATCC 43504 was the most tolerant. Among the bacteriocins tested, lacticins A164 and BH5 produced by Lactococcus lactis subsp. lactis A164 and L. lactis BH5, respectively, showed the strongest antibacterial activity against H. pylori strains. MICs of the lacticins against H. pylori strains, when assessed by the critical dilution micromethod, ranged from 0.097 to 0.390 mg/liter (DSM strains) or from 12.5 to 25 mg/liter (ATCC 43504), supporting the strain-dependent sensitivity of the pathogen. Pediocin PO2 was less active than the lacticins against four strains of H. pylori, and leucocin K was the least active peptide, with no inhibition toward H. pylori ATCC 43504. Anti-Helicobacter activity of lacticin A164 was dependent on initial inoculum size as well as concentration of the bacteriocin added.     

Factors influencing the production of acetoin and diacetyl by lactic acid bacteria in skim milk

Nine strains of starter cultures belonging to the genera Streptococcus, Leuconostoc and Lactobacillus were studied for the effect of pyruvate concentration, incubation period and the associative growth on the production of acetoin and diacetyl in skim milk. The absolute amount of acetoin and diacetyl and the amount pro μmol pyruvate increased with increasing the concentration of pyruvate. At the low concentration of pyruvate the maximum amount of these neutral carbonyl compounds was first reached after 34 days, whereas at the high concentration it was readily achieved after 6 days and then decreased. Production of acetoin and diacetyl by mixed-species cultures from added citrate was either increased or markedly decreased compared to the single-species culture.     

乳酸菌发酵代谢合成叶酸的影响因素

对嗜酸乳杆菌以及乳酸乳球菌发酵合成叶酸的影响因素进行了研究。结果表明,乳酸菌代谢合成叶酸的产率为17~100μg/L,菌种、培养时间、pH值、对氨基苯甲酸(PABA)质量浓度会影响乳酸菌合成叶酸的产量。与乳酸乳球菌乳酸亚种相比,嗜酸乳杆菌CH-2生成的叶酸产量要高。不同菌株生成叶酸的能力与pH值有关,嗜酸乳杆菌在pH值为4.2叶酸产率明显下降,乳酸乳球菌乳酸亚种产叶酸的能力则不受pH值影响。添加PABA可以显著提高乳酸菌的叶酸产率。选择适宜的乳酸菌菌株,优化发酵工艺参数可以提高乳及相关食品中叶酸的质量浓度,达到生物方式强化叶酸的效果。     

Nutritional factors affecting the production of two bacteriocins from lactic acid bacteria on whey

The ability of Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 to produce bacteriocins on both diluted and concentrated whey was investigated in batch fermentations. Both strains produced the higher amounts of biomass and bacteriocin titres on diluted whey. Luedeking and Piret expression was able to model the production of nisin, which was produced as a primary metabolite on both culture media. However, the pediocin production could not be typified in any case due to the negligible growth of P. acidilactici. Although the whey supported the growth and bacteriocin production by the two strains, both biomass and bacteriocin productions were lower than those obtained on MRS broth. The effect of total sugar, nitrogen, phosphorous and buffer concentrations on the production of nisin and pediocin was studied in diluted whey using factorial experiments and empirical modelling. The production of nisin was greatly inhibited by the increase in nitrogen, buffer, and to a lesser extent, sugar concentration in the medium, nevertheless, the used phosphorous source produced a light stimulatory effect on bacteriocin synthesis. In addition, the growth of Lc 1.04 was mainly affected by the nitrogen source used. On the other hand, pediocin was inhibited by the increase in buffer, phosphorous, and to a lesser degree, by the sugar and nitrogen concentration.

The inhibitory activity of pediocin disappeared almost totally after 15 min of treatment with trypsin, papain, subtilisin and pepsin. The activity of nisin was drastically reduced by treatment with trypsin, subtilisin and pepsin. Nevertheless, 50% of the initial activity was retained when nisin was treated with papain. Both bacteriocins showed the highest heat stability at acidic pH and short incubation times.     

Diversity of bacteriocins produced by lactic acid bacteria isolated from raw milk

Bacteriocin-producing lactic acid bacteria were isolated from 298 samples of raw ewes', goats’ or cows’ milk. Eighty-two bacteriocin producers were phenotypically and genotypically identified as L. lactis subsp. lactis (59 isolates), L. lactis subsp. cremoris (2 isolates), L. lactis subsp. lactis biovar diacetylactis (6 isolates), E. faecalis (7 isolates), E. faecium (1 isolate), L. paracasei subsp. paracasei (4 isolates), L. plantarum (1 isolate) and Leuconostoc spp. (2 isolates). By means of PCR-techniques, nisin was characterized in 39 of the 67 bacteriocin-producing lactococci and lacticin 481 in 23 isolates, some of which presented antilisterial activity. Enterocin AS-48 was produced by four enterococcal isolates. Four non-identified bacteriocins produced by 16 isolates showed a broad inhibitory spectrum. Nisin-producing lactic acid bacteria were the most abundant, but lacticin 481-producing lactococci and AS-48-producing enterococci were found at relatively high rates.     

Growth-inhibition of hiochi bacteria in namazake (raw sake) by bacteriocins from lactic acid bacteria

The bacteriocins produced by Lactococcus lactis subsp. lactis C101910 (C101910) and NBRC 12007 (NBRC 12007) were used to prevent the growth of sake spoiling hiochi bacteria (Lactobacillus hilgardii, Lactobacillus fructivorans, and Lactobacillus paracasei) in namazake, which is raw (unpasteurized) sake. The bacteriocin concentrations required for decreasing the viable cell concentrations of L. hilgardii and L. fructivorans below the detection limit (1.0 × 10² cells/ml) in 24 h from the initial concentration of 4.0–9.5 × 10⁵ cells/ml in the namazake at pH 4.5 and at 4°C, were 18–35 U/ml and 5.6 U/ml for the bacteriocin from C101910 and NBRC 12007, respectively. To decrease the viable cell concentration of L. paracasei from the initial concentration of 7.5 × 10⁵ cells/ml to below the detection limit (1.0 × 10² cells/ml) in 24 h, 350 U/ml bacteriocin from C101910 and 140 U/ml bacteriocin from NBRC 12007 were required. In experiments using McIlvaine buffer (pH 4.5) with 15% ethanol instead of namazake as the medium, the viable cell concentrations of L. hilgardii and L. paracasei decreased to less than 1.0 × 10² cells/ml, whereas those of L. fructivorans decreased to less than 1.0 × 10³ cells/ml, when bacteriocins were added at the concentrations that had proven effective in namazake. The membrane depolarization assay using a fluorescent probe showed that the presence of ethanol stimulated the collapse of the membrane potential induced by bacteriocins. The ethanol induced collapse of the membrane potential suggests that the application of bacteriocins at the storage stage of namazake is more beneficial than when used in other stages of the sake brewing process.     

PCR-DGGE analysis of population dynamics of lactic acid bacteria in kimchi by addition of bacteriocins

Food Science and Biotechnology - The effects of two crude bacteriocins (DF01 and K10) on lactic acid bacteria (LAB) communities and pH during kimchi fermentation were analyzed by polymerase chain...     

乳酸菌Ⅱ类细菌素生物合成及其代谢调控策略

乳酸菌细菌素是乳酸菌经由核糖体合成的具有抑菌活性的多肽类物质,其中乳酸菌Ⅱ类细菌素具有很强的热稳定性和抑菌活性,但是产量较低尚不能实现商业化生产。因此,了解和掌握乳酸菌Ⅱ类细菌素合成及其代谢调控机制,对解决商业化生产过程的技术瓶颈具有重要的意义。该文综述了目前Ⅱ类细菌素生物合成和降解途径,并从代谢调控角度提出提高Ⅱ类细菌素产量的可行性策略,为提高Ⅱ类细菌素产量提供新的途径和思路,也为Ⅱ类细菌素工业化生产奠定基础。     

Combating biofilms of foodborne pathogens with bacteriocins by lactic acid bacteria in the food industry

Most foodborne pathogens have biofilm-forming capacity and prefer to grow in the form of biofilms. Presence of biofilms on food contact surfaces can lead to persistence of pathogens and the recurrent cross-contamination of food products, resulting in serious problems associated with food safety and economic losses. Resistance of biofilm cells to conventional sanitizers urges the development of natural alternatives to effectively inhibit biofilm formation and eradicate preformed biofilms. Lactic acid bacteria (LAB) produce bacteriocins which are ribosomally synthesized antimicrobial peptides, providing a great source of nature antimicrobials with the advantages of green and safe properties. Studies on biofilm control by newly identified bacteriocins are increasing, targeting primarily onListeria monocytogenes, Staphylococcus aureus, Salmonella, and Escherichia coli. This review systematically complies and assesses the antibiofilm property of LAB bacteriocins in controlling foodborne bacterial-biofilms on food contact surfaces. The bacteriocin-producing LAB genera/species, test method (inhibition and eradication), activity spectrum and surfaces are discussed, and the antibiofilm mechanisms are also argued. The findings indicate that bacteriocins can effectively inhibit biofilm formation in a dose-dependent manner, but are difficult to disrupt preformed biofilms. Synergistic combination with other antimicrobials, incorporation in nanoconjugates and implementation of bioengineering can help to strengthen their antibiofilm activity. This review provides an overview of the potential and application of LAB bacteriocins in combating bacterial biofilms in food processing environments, assisting in the development and widespread use of bacteriocin as a promising antibiofilm-agent in food industries.     

乳酸菌细菌素的高效表达方法研究

乳酸菌细菌素作为天然的生物防腐剂,因其高效、无毒等特点而备受关注,但目前只有乳酸链球菌素实现了工业化生产。乳酸菌细菌素产量低是限制其大规模生产的瓶颈之一。从高产菌株筛选、发酵条件调控、基因工程技术、诱导调控和胁迫刺激应答5个方面,综述了提高乳酸菌细菌素产量的方法,期望为提高乳酸菌细菌素产量的研究提供新思路。     

A rapid turbidometric microplate bioassay for accurate quantification of lactic acid bacteria bacteriocins

A 1 day turbidometric microplate bioassay (TMB) was developed for the rapid, accurate and precise quantification of lactic acid bacteria (LAB) bacteriocins (nisin Z and pediocin PA-1). Parameters such as the concentration of the indicator strains and the incubation time were optimized for each bacteriocin. A high correlation coefficient (r²=0.992±0.004) was obtained for the exponential regression in the nisin Z concentration range of 20–120 ng/ml with 1×10⁷ CFU indicator strain (Pediococcus acidilactici UL5) and an incubation time of 3 h. Using these parameters, the detection limit was estimated at 80 ng/ml (3.2 IU/ml), compared to 300 ng/ml for the agar diffusion assay (ADA). High precision (<7%) and accuracy (10%) were obtained for all nisin Z concentrations tested. Similar results were obtained with pediocin PA-1 with r²=0.993±0.005, a precision (8.2%) and an accuracy lower than 15%.     

Factors affecting capsule size and production by lactic acid bacteria used as dairy starter cultures

The effects of sugar substrates on capsule size and production by some capsule-forming nonropy and ropy dairy starter cultures were studied. Test sugars (glucose, lactose, galactose, or sucrose) were used as a sole carbohydrate source and the presence of a capsule and its size were determined by using confocal scanning laser microscopy. Nonropy strains produced maximum capsule size when grown in milk. Strains that did not produce capsules in milk did not produce them in any other growth medium. Specific sugars required for capsule production were strain-dependent. Increasing lactose content of Elliker broth from 0.5 to 5% or adding whey protein or casein digest produced larger capsules. Whey protein concentrate stimulated production of larger capsules than did casamino acids or casitone. Some Streptococcus thermophilus strains produced capsules when grown on galactose only. Nonropy strains of Lactobacillus delbrueckii subsp. bulgaricus produced capsules on lactose, but not on glucose. A ropy strain of Lactobacillus delbrueckii subsp. bulgaricus produced a constant capsule size regardless of the growth medium. The ability of some strains of Streptococcus thermophilus to use galactose in capsule production could reduce browning of mozzarella cheese during baking by removing a source of reducing sugar. Media that do not support capsule production may improve cell harvesting.     

Class IIa bacteriocins from lactic acid bacteria: antibacterial activity and food preservation

In the last decade, a variety of ribosomally synthesized antimicrobial peptides, or bacteriocins, produced by lactic acid bacteria have been identified and characterized. As a result of these studies, insight has been gained into various fundamental aspects of biology and biochemistry such as bacteriocin processing and secretion, mechanisms of cell immunity, and structure-function relationships. In parallel, there has been a growing awareness that bacteriocins may be developed into useful antimicrobial food additives. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their significant biological activities and potential applications. The present review provides an overview of the knowledge available for class IIa bacteriocins and discusses common features and recent findings concerning these substances. The activity and potential food applications of class IIa bacteriocins are a major focus of this review.     

乳酸菌细菌素研究进展及其在肉制品防腐保鲜领域的应用

乳酸菌细菌素是一种天然的生物防腐剂,可以抑制或杀灭食品里致病微生物和腐败微生物从而保证食品安全。文中概述了乳酸菌细菌素的分类、作用机制、分离纯化方法及在肉制品防腐保鲜领域的应用,并展望了以后的研究方向。     

Citrulline production by lactic acid bacteria in Chinese rice wine

Ethyl carbamate (EC) is a carcinogenic compound derived from the spontaneous reaction of ethanol with urea or citrulline in Chinese rice wine. Polymerase chain reaction–denaturing gradient gel electrophoresis showed that five species, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis and Lactobacillus coryniformis were the most abundant bacteria in the Chinese rice wine production process. Five strains belonging to these species can degrade arginine primarily in the exponential growth phase and accumulate citrulline in MRS‐Arg medium. In addition, an L. brevis strain was shown to be capable of assimilating citrulline, indicating the potential of this strain suggesting a potential route to reduce citrulline content and ethyl carbamate formation in Chinese rice wine fermentation. Copyright © 2018 The Institute of Brewing & Distilling     

废蜜制取乳酸新菌种

回顾了利用糖蜜等有机化合物进行乳酸生产的技术和菌种研究进展。发酵工业上应用最多的生产乳酸的原料是葡萄糖、蔗糖、淀粉等糖类物质，虽然用纯糖物质发酵得到的乳酸产率高，提取也相对容易，但用高价的糖生产低价格的乳酸在经济上是不合算的，且还浪费了资源。利用含丰富碳水化合物的有机废弃物发酵生产乳酸，不但可以解决废弃物的资源化问题，还能降低乳酸的生产成本，具有广阔的开发前景，甜菜废蜜就是一种可以利用的底物。     

The effects on vegetative cells and spores of three bacteriocins from lactic acid bacteria

The sensitivities of vegetative cells of strains ofListeria, Clostridium, Staphylococcus, Lactococcus, Lactobacillus, MicrococcusandPediococcus, and of spores ofClostridiumandBacillusto three broad spectrum bacteriocins (nisin A, nisin Z and pediocin) from lactic acid bacteria were determined by a critical dilution micro-assay. The minimal inhibitory concentrations (MIC) of partially purified bacteriocins, prepared by a pH-dependent adsorption/desorption process, were determined and expressed in arbitrary units ml⁻¹and in μ g ml⁻¹of pure bacteriocin. The MICs of bacteriocins varied considerably between species and even between strains of the same species, as clearly shown for nine strains ofListeria monocytogenes. When bacteriocin activity was expressed in μ g ml⁻¹, pediocin was more effective againstListeria monocytogenesthan nisin A or nisin Z. The latter bacteriocins, in concentrations between 23 and 69 μ g ml⁻¹, prevented outgrowth ofClostridiumandBacillusspores for at least 10 days. Although pediocin at 17 μ g ml⁻¹prevented outgrowth ofB. stearothermophilusandC. butyricumspores for up to 7 days, it apparently activated the germination ofB. subtilisspores.