Effect of enterocin AS-48 in combination with biocides on planktonic and sessile Listeria monocytogenes

Enterocin AS-48 was tested on a cocktail of Listeria monocytogenes strains in planktonic and sessile states, singly or in combination with biocides benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide, triclosan, poly-(hexamethylen guanidinium) hydrochloride, chlorhexidine, hexachlorophene, and the commercial sanitizers P3 oxonia and P3 topax 66. Combinations of sub-inhibitory bacteriocin concentrations and biocide concentrations 4 to 10-fold lower than their minimum inhibitory concentrations (MIC) completely inhibited growth of the planktonic listeriae. Inactivation of Listeria in biofilms formed on polystyrene microtiter plates required concentrations of enterocin AS-48 greater than 50 μg/ml, and biocide concentrations ten to 100-fold higher. In combination with enterocin AS-48 (25 or 50 μg/ml), microbial inactivation increased remarkably for all biocides except P3 oxonia and P3 topax 66 solutions. Polystyrene microtiter plates conditioned with enterocin solutions (0.5-25 μg/ml) decreased the adherence and biofilm formation of the L. monocytogenes cell cocktail, avoiding biofilm formation for at least 24 h at a bacteriocin concentration of 25 μg/ml.     

Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin

A microtiter plate assay was employed to systematically assess the interaction between ethylenediaminetetraacetic acid (EDTA) or lactoferrin and nisin, lysozyme, or monolaurin against strains of Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, and Pseudomonas fluorescens. Low levels of EDTA acted synergistically with nisin and lysozyme against L. monocytogenes but EDTA and monolaurin interacted additively against this microorganism. EDTA synergistically enhanced the activity of nisin, monolaurin, and lysozyme in tryptic soy broth (TSB) against two enterohemorrhagic E. coli strains. In addition, various combinations of nisin, lysozyme, and monolaurin with EDTA were bactericidal to some gram-negative bacteria whereas none of the antimicrobials alone were bactericidal. Lactoferrin alone (2000 microg ml(-1)) did not inhibit any of the bacterial strains, but did enhance nisin activity against both L. monocytogenes strains. Lactoferrin in combination with monolaurin inhibited growth of E. coli O157:H7 but not E. coli O104:H21. While lactoferrin combined with nisin or monolaurin did not completely inhibit growth of the gram-negative bacteria, there was some growth inhibition. All combinations of EDTA or lactoferrin with antimicrobials were less effective in 2% fat UHT milk than in TSB. S. enteritidis and P. fluorescens strains were consistently more resistant to antimicrobial combinations. Resistance may be due to differences in the outer membrane and/or LPS structure.     

Encapsulation of nisin and lysozyme in liposomes enhances efficacy against Listeria monocytogenes

The efficacy and stability against Listeria monocytogenes of nisin and lysozyme encapsulated in phospholipid liposomes was evaluated. Antimicrobial-containing liposomes were prepared by hydrating dried lipids with buffer containing nisin, nisin plus the fluorescence probe calcein, or calcein and lysozyme. Mixtures were then centrifuged and sonicated, and encapsulated liposomes were collected using size-exclusion chromatography. Antimicrobial concentration in liposomes was determined by bicinchoninic acid assay prior to determination of antimicrobial activity against strains of L. monocytogenes. When nisin was encapsulated in liposomes, protein concentrations of 0.39, 0.27, and 0.23 mg/ml for phosphatidylcholine (PC), PC-cholesterol (7:3), and PC-phosphatidylglycerol (PG)-cholesterol (5:2:3), respectively, were obtained. Encapsulation of nisin with calcein yielded protein concentrations of 0.35, 0.39, and 0.28 mg/ml for PC, PC-cholesterol, and PC-PG-cholesterol, respectively. Encapsulation of calcein with lysozyme resulted in protein concentrations of 0.43, 0.26, and 0.19 mg/ml for PC, PC-cholesterol, and PC-PG-cholesterol, respectively. Encapsulated nisin in 100% PC and PC-cholesterol liposomes inhibited bacterial growth by >2 log CFU/ml compared with free nisin. Growth inhibition with liposomal lysozyme was strain dependent, with greater inhibition observed for strains 310 and Scott A with PC-cholesterol and PC-PG-cholesterol liposomes. Inhibition of L. monocytogenes indicated the potential of liposomes to serve as delivery vehicles for antimicrobials in foods while improving stability of antimicrobials.     

Specific Bifidobacterium strains isolated from elderly subjects inhibit growth of Staphylococcus aureus

Cell-free, pH-controlled supernatants of thirty-eight Bifidobacterium strains isolated from healthy elderly subjects were subjected to antimicrobial activity assay. Bioluminescent indicator strains Staphylococcus aureus RN4220, Escherichia coli K-12, and Salmonella enterica serovar Typhimurium ATCC 14028 were used as targets of antimicrobial activity. The effect of nutrient depletion on the inhibition was eliminated with spent-culture controls. Three out of thirty-eight Bifidobacterium strains were capable of inhibiting the growth of S. aureus. The inhibition was equal to 23.2+/-19.1% to 50.4+/-26.7% of the inhibition caused by 50 IU/ml nisin. Reuterin-producing positive strain Lactobacillus reuteri SD2112 was capable of 86.0+/-24.6% inhibition, but Bifidobacterium lactis Bb-12, a known probiotic strain, showed no inhibition. None of the strains was capable of inhibiting the growth of E. coli or S. enterica. The observed inhibition by bifidobacteria was related to hydrogen peroxide formation and possible production of heat-stable proteinaceous compounds. The results suggest that production of antimicrobial substances other than organic acids is not common among Bifidobacterium strains typical of elderly subjects. However, specific strains were identified which showed considerable inhibitory activity against S. aureus.     

Impedance measurement of growth of lactic acid bacteria in the presence of nisin and lysozyme

Effect of nisin (0.500 g/l), lysozyme (0.035 g/l) and the mix of nisin (0.500 g/l) and lysozyme (0.035 g/l) against two nisin-producing lactococci strains, three non-nisin-producing lactococci strains and four lactobacilli strains with antimicrobial activity was determined by the impedimetric method. None of the tested lactococci and lactobacilli strains were inhibited by lysozyme at concentration of 0.035 g/l. Only the nisin-producing lactococci strains were resistant to nisin at 0.500 g/l, the other lactococci and lactobacilli were inhibited by nisin at 0.500 g/l intensively. The mix of lysozyme and nisin displayed a comparable effect to the nisin addition against the used lactococci strains and was very variable depending on the strain of tested lactobacilli. The impedimetric method was shown to be convenient for screening the growth abilities of lactococci and lactobacilli in the presence of antimicrobial components.     

乳酸链球菌素与L-乳酸对嗜水气单胞菌的协同抑杀作用

为探讨乳酸链球菌素(Nisin)与L-乳酸对嗜水气单胞菌的协同抑杀作用,分别采用比浊法、活菌计数法、膜电动势探针等检测Nisin与L-乳酸对嗜水气单胞菌(ATCC 35654、CICC 10500两株菌)生长、存活、生物被膜形成以及菌体膜完整性的影响。结果表明,Nisin与低浓度(3.5 mmol/L)L-乳酸协同,显著抑制了两株嗜水气单胞菌的生长,使生长延滞期延长,最大比生长速率下降,成熟期最高活菌数量降低,且抑制效果随Nisin浓度增加而增强。Nisin和L-乳酸对指示菌有协同致死作用,Nisin与L-乳酸协同处理12 h,可显著(P<0.05)降低嗜水气单胞菌活菌数,造成菌体细胞死亡。机理初探表明:低浓度L-乳酸可以使菌株外膜脂多糖(Lipopolysaccharide,LPS)释放,Nisin进一步使菌体膜电动势消散。研究结果为利用Nisin与L-乳酸或二者的产生菌控制水产品中的嗜水气单胞菌提供了理论依据。     

乳链菌肽的抑菌机制

乳链菌肽(Nisin)是由乳酸乳球菌的某些菌株产生的1种羊毛硫细菌素。因其高效的抗菌性及对人体的低毒性而被广泛应用于食品防腐。孔道形成是Nisin抑菌作用的主要方式。Nisin可通过其带正电荷肽的C-端与带负电荷的磷脂之间的静电引力在细胞质膜上形成孔道,也能通过lipid Ⅱ的锚定作用形成lipid Ⅱ介导的孔道,同时Nisin与lipid Ⅱ结合抑制肽聚糖生物合成。Nisin对大肠杆菌的抑菌机制是对高盐敏感,而其对葡萄球菌的抑菌机制是对高盐不敏感。文章综述了Nisin的抑菌机制。     

Evaluation of the ability of lysozyme and nisin to control meat spoilage bacteria.

The antimicrobials lysozyme, nisin, and mixtures of the two were studied to ascertain their abilities to control the growth of the meat-borne spoilage bacteria, Brochothrix thermosphacta B2 and Carnobacterium sp. 845. The goal was to optimize an antimicrobial for potential use in preservation of fresh meats. Their efficacies were evaluated in APT broth, in a meat juice extract and on cores of lean and fat pork tissue. Both lysozyme and nisin alone as well as mixtures of the two effectively inhibited B. thermosphacta B2 at 250 microg/ml in APT broth, the lowest concentration evaluated, for 10 days at 2 degrees C. In the presence of 500 microg/ml lysozyme, B. thermosphacta B2 grew after 12 days incubation. Only 125 microg of antimicrobial/ml was required to inhibit B. thermosphacta B2 for 27 days at 2 degrees C in pork juice. An estimated surface concentration of 130 microg/cm2 of each of the antimicrobials effectively inhibited B. thermosphacta B2 on inoculated cores of fat and lean pork tissue when the cores were incubated in vacuum packages for 6 weeks at 2 degrees C. In APT broth and in pork juice, lysozyme showed no antimicrobial activity against Carnobacterium sp. 845 at concentrations of 500 and 1000 microg/ml, respectively. Nisin and mixtures of the two antimicrobials inhibited Carnobacterium sp. 845 so that its numbers were at least 3 log units lower than untreated samples after 26 and 27 days incubation for APT and pork juice, respectively. The antimicrobial effect was concentration dependent. On lean pork tissue, numbers of Carnobacterium sp. 845 were significantly lower than untreated samples or samples treated with 195 microg/cm2 lysozyme when 260 microg/cm2 of a 1:3 (w/w) ratio of nisin to lysozyme was introduced to the cores. The inhibitory effect lasted for 14 of 42 days incubation in vacuum at 2 degrees C. On fat tissue, both lysozyme alone and the 1:3 nisin/lysozyme mixture inhibited Carnobacterium sp. 845 for 21 days storage in vacuum at 2 degrees C. On fat and lean tissue, mixtures of nisin and lysozyme would be more effective antimicrobials than either nisin or lysozyme alone.     

Efficacy of the Antimicrobial Peptide Nisin in Emulsifying Oil in Water

Nisin is an amphiphilic, antimicrobial peptide that has found application as a preservative in the food industry. Evaluation of nisin as an emulsifier was accomplished by measuring its ability to stabilize dispersed oil droplets in water. This was achieved by monitoring the time-dependent electrical conductivity of the dispersion. Nisin solutions at concentrations of 0.1, 0.5, 1, 5, and 10 mg/ml were used to prepare emulsions, by homogenization of a volume of nisin solution and corn oil. While nisin showed significant emulsifying capacity in comparison to Tween® 80 and β-casein, its emulsifying properties were found to be highly concentration- and pH-dependent.     

培养基与分割发酵优化促进Nisin生物合成

为了提高乳酸乳球菌（Lactococcus lactis）细胞生长和乳酸链球菌素（Nisin）的合成效率,以正交优化法研究培养基分批发酵和分割发酵方式对Nisin生物合成效率的影响。结果表明,优化发酵培养基配方为：5%蛋白胨,3%蔗糖,2%玉米浆,1%酵母浸粉。在此优化条件下,摇瓶培养（11 h）峰值生物量为4.9×109 CFU/mL,较对照提高43%;10 L发酵罐分批发酵峰值生物量、Nisin效价及Nisin合成速率■q分别为7.75×109 CFU/mL、2 573 IU/mL、151.4 IU/（mL·h）,较优化前分别提升38.0%、56.6%、38.2%。分批发酵培养18 h Nisin达到峰值后[■q为147 IU/（mL·h）],以不同比例分割发酵并继续培养7 h,第二次分割（25%）为Nisin合成最适发酵方式,其Nisin平均合成速率达到294 IU/（mL·h）,较分批发酵提高了100%。     

NISIN: A BACTERIOCIN WITH A POTENTIAL USE IN BREWING

Depending on the amount used and the strain of bacteria involved, nisin either kills lactic acid bacteria or inhibits their growth. In medium inoculated with approximately 10⁵ cells ml^?1 of a sensitive strain of Lactobacillus (BSO 375) nisin, added at levels recommended for commercial use (100 International Units ml^?1, killed all the cells in less than 6 h. In the absence of nisin this inoculum grew to a concentration of 10¹⁰ cells ml^?1 in about 50 h. Lower nisin concentrations killed fewer cells but inhibited the growth of those still viable. For the more resistant strain Lactobacillus (BSO 343) growth was only inhibited at the higher nisin concentrations. Nisin maintained its activity against lactic acid bacteria in brewing fermentations. It had no effect on the growth and fermentative performance of the 9 brewing yeast strains tested, and, in a pilot brewery fermentation, had no deleterious affect on the taste of the beer produced. Nisin could be used either as a preventative measure by regular addition to fermentations, or as a remedial measure once contamination by lactic acid bacteria had been detected.     

Possible synergistic effect of nisin and propionic acid on the growth of the mycotoxigenic fungi Aspergillus parasiticus, Aspergillus ochraceus, and Fusarium moniliforme.

The effects of nisin and propionic acid (PA) on aflatoxin production and on mycelial growth and spore germination of the mycotoxigenic fungi Aspergillus parasiticus, A. ochraceus, and Fusarium moniliforme were investigated. The growth of A. ochraceus was completely inhibited on media containing PA with nisin in concentrations of 0.05% PA with 1,000 ppm nisin, and 0.1% PA with 500 or 1,000 ppm nisin. The growth of both F. moniliforme and A. parasiticus was completely inhibited by PA with nisin at a concentration of 0.1% PA with 1,000 ppm nisin. Nisin alone caused a significant increase in mycelial growth when applied to A. ochraceus at 500 or 1,000 ppm and when applied to A. parasiticus at 1,000 ppm. Spore germination of A. ochraceus was completely inhibited on media containing 0.1% PA with 500 or 1,000 ppm nisin. Spores of F. moniliforme failed to germinate in 0.05% PA with 500 or 1,000 ppm nisin, whereas spores of A. parasiticus did not germinate on media containing 0.1% PA with 1,000 ppm nisin. For all three fungi tested, the inhibitory effect on mycelial growth was found to be fungistatic rather than fungicidal. The combined treatment of PA with nisin produced better fungistatic activity than treatment involving either material alone. Nisin, applied alone, did not stimulate aflatoxin production (expressed by microg toxin/mg mycelium), but the combined treatment at certain concentrations was inhibitory to aflatoxin B1 or G1. The production of aflatoxin G1, but not of B1, was stimulated in 0.05% PA with 1,000 ppm nisin and on media containing 0.1% PA with 100 ppm nisin. Nisin is currently applied in foods to prevent spoilage induced by bacteria but not by mold. The results of the present study indicate that a combined treatment of nisin in small concentrations of PA might be useful in preventing mold damage in certain foods and stored grain.     

广东地区46株食源性金黄色葡萄球菌耐药性、生物被膜形成能力及IcaAB基因的特征分析

目的 分析广东地区食源性金黄色葡萄球菌耐药现状、生物被膜形成能力及生物被膜相关基因IcaAB的特征。方法 对46株食源性金黄色葡萄球菌采用微量肉汤稀释法进行药敏实验,采用结晶紫法检测生物被膜生成能力,运用聚合酶链式反应(polymerase chain reaction, PCR)检测生物被膜相关基因。结果 97.83%的菌株对至少1种抗生素耐药, 30.43%为多重耐药菌株,对青霉素(97.83%)、氨苄西林(97.83%)耐药性最高; 65.22%菌株能够形成生物被膜,其中强、中和弱粘附生物被膜能力的菌株分别占17.39%、30.43%和17.39%,且产生物被膜能力强的菌株对特定抗生素(头孢西丁、环丙沙星)的耐药性更高(P<0.05); PCR结果显示, icaA、icaB的检出率分别为56.67%、36.67%,基因携带率与产膜能力无显著关联(P>0.05)。结论 广东地区食源性金黄色葡萄球菌普遍表现出耐药性和生物被膜形成能力,对公共卫生带来挑战,需采用措施以确保公共安全。     

Antimicrobial edible packaging based on cellulosic ethers, fatty acids, and nisin incorporation to inhibit Listeria innocua and Staphylococcus aureus

Edible cellulosic films made with hydroxypropylmethylcellulose (HPMC) have proven to be inadequate moisture barriers. To improve its water vapor barrier properties, different hydrophobic compounds were incorporated into the HPMC matrix. Some fatty acids and derivatives were included into the film-forming solution prior to film formation. Stearic acid was chosen because of its high capacity to reduce significantly the water vapor transmission rate. Antimicrobial activity of edible HPMC film was obtained by the incorporation of nisin into the film-forming solution. Nisin is an antimicrobial peptide effective against gram-positive bacteria. The inhibitory activity of this bacteriocin was tested for inhibition of Listeria innocua and Staphylococcus aureus. The use of stearic acid was observed to reduce the inhibitory activity of active HPMC film against both selected strains. This phenomenon may be explained by electrostatic interactions between the cationic nisin and the anionic stearic acid. Further studies showed that antimicrobial activity of film varied with the nature of the hydrophobic compound incorporated, in decreasing order: film without lipid, methylstearate film, and stearic acid film. This corroborated the idea of electrostatic interactions.     

The effect of nisin on the physiology of Bifidobacterium thermophilum

The effects of nisin on lactate accumulation, growth, and Fe(III) binding by Bifidobacterium thermophilum (ATCC 25866) and Bifidobacterium breve (ATCC 15700) were investigated. Nisin inhibited lactate production by B. thermophilum at concentrations of less than 1 microg/ml, but this effect could be largely eliminated by pretreatment of the organism with 100 to 400 microM Al(III) or La(III). Nisin also inhibited the growth of B. thermophilum at concentrations of 2 to 3 microg/ml, with lower concentrations showing lag periods and/or slower rates of growth. However, Al(III) could not negate these effects, most likely because of Al(III) chelation by the trypticase-proteose-yeast extract medium. Nisin was able to increase instantaneous Fe(III) binding by both B. thermophilum and B. breve, though prolonged-time experiments (up to 120 min) with B. thermophilum indicated no difference in total Fe(III) bound. Nisin was thus able to increase the free radical reaction rate with bifidobacteria and the resultant rate of Fe(III) binding. It was concluded that nisin will normally inhibit the metabolic activity of B. thermophilum along with that of certain bacterial pathogens; however, this effect may in some instances, be abated by a pretreatment with Al(III). Moreover, by accelerating free radical action and the binding of iron by bifidobacteria, nisin may be able to potentiate their normal probiotic action.     

Inhibition of Bacillus anthracis and potential surrogate bacilli growth from spore inocula by nisin and other antimicrobial peptides

The ability of nisin, synthetic temporin analogs, magainins, defensins, and cecropins to inhibit Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus subtilis growth from spore inocula was determined using well diffusion assays. Nisin, magainin II amide, and defensins were inhibitory in screening against B. anthracis Sterne or B. cereus ATCC 7004, but only nisin inhibited virulent B. anthracis strains. The MICs of nisin against the 10 Bacillus strains examined were 0.70 to 13.51 microg/ml. Synthetic temporin analogs also inhibited B. anthracis but were not as potent as nisin. None of the strains examined were appropriate B. anthracis surrogates for testing sensitivity to antimicrobial peptides.     

Inhibition of Listeria innocua in hummus by a combination of nisin and citric acid

The effect of nisin or citric acid or combinations of these two inhibitors on the inactivation of a cocktail of three Listeria innocua strains was investigated in a model brain heart infusion (BHI) broth and hummus (chickpea dip). In BHI broth, citric acid had a limited ability to inhibit L. innocua growth. Nisin initially reduced L. innocua concentrations by about 3 log cycles; however, L. innocua reached concentrations similar to those of the control after 5 days at 22 degrees C. In combination, the effects of 500 IU/ml nisin and 0.2% citric acid were synergistic and resulted in complete elimination of L. innocua in the BHI broth. The inhibition of L. innocua by nisin (500 or 1,000 IU/g), citric acid (0.1, 0.2, or 0.3%), or their combinations also was evaluated in hummus. Citric acid alone did not affect L. innocua growth or the aerobic bacterial plate count. A combination of 1,000 IU/g nisin and 0.3% citric acid was somewhat effective (approximately 1.5-log reduction) in controlling the concentration of L. innocua and the aerobic plate count for up to 6 days. This combination also may be useful, in addition to proper hygienic practices, for minimizing the growth of the pathogen Listeria monocytogenes in hummus.     

Effective use of nisin to control lactic acid bacterial spoilage in vacuum-packed bologna-type sausage.

Lactic acid bacteria (LAB) commonly cause spoilage in minimal heat-treated vacuum-packed cured delicatessen meats. Predominant species are Lactobacillus sake and L. curvatus. LAB strains isolated from spoiled products of this type (liver sausage, ham and bologna sausage) were found to be sensitive to low nisin concentrations (maximum of 1.25 microg g(-1)). Addition of 25 microg g(-1) nisin (as Nisaplin) inhibited the growth of LAB spoilage organisms inoculated into vacuum-packed pasteurized bologna-type sausages stored at 8 degrees C. Control sausages became spoiled (>10(8) LAB CFU g(-1)) by day 7, whereas sausages containing nisin remained unspoiled for >50 days. The effect of three types of phosphates (used as emulsifiers) on nisin activity in the sausages was compared. LAB growth rate was fastest in samples containing orthophosphate, and slowest in sausages containing diphosphate. The shelf life was also greatly extended in the latter. Fat content also affected nisin activity. Nisin activity (as indicated by LAB inhibition) was greatest in samples containing 15% > 25% > 37% (wt/wt) fat. In a sausage formulation containing 37% fat and incorporating diphosphate as emulsifier, levels of nisin as low as 2.5 microg g(-1) showed antibacterial effects. A nisin level of 6.25 microg g(-1) totally inhibited LAB growth for over 4 weeks and 25 microg g(-1) for 5 weeks. Spoilage control was achieved in the same sausage formulation but with 25% (wt/wt) fat; 12.5 microg g(-1) nisin prevented LAB growth for 5 weeks.     

Effect of Nisin on the Outgrowth of Clostridium botulinum Spores

Nisin, an antibiotic produced by certain strains of Streptococcus lactis, is effective in preventing the outgrowth of Clostridium botulinum spores. Type A C. botulinum spores were the most resistant to the inhibitory action of nisin requiring 1000-2000 I.U. of nisin/ml for a 50% inhibition of outgrowth on TPYG agar plates. Type E spores were more sensitive requiring only 50-100 I.U./ml for 50% inhibition of outgrowth on TPYG agar plates. Type B spores displayed an intermediate level of sensitivity requiring 500-1000 I.U. of nisin/ml for 50% inhibition of outgrowth on TPYG agar plates. Similar levels of nisin were necessary to prevent spore outgrowth in TPYG broth and BHI broth over a 7-day incubation period. With prolonged incubation periods of up to 65 days in TPYG broth, spore outgrowth was observed sporadically at higher nisin levels with the type A and B spores which may indicate some decomposition of nisin with storage. Nisin levels of 5000 I.U./ml for the type A spores and 2000 I.U./ml for the type B spores and the Minnesota E spores were insufficient to prevent spore outgrowth by C. botulinurn in cooked meat medium. For the Beluga E spores, a nisin level of 2000 I.U./ml was necessary to prevent spore outgrowth in cooked meat medium. The need for higher levels of nisin in cooked medium to prevent spore outgrowth may be due to the binding of the nisin by meat particles.     

乳链菌肽发酵条件的研究

利用分批培养，对乳酸链球菌ＳＭ５２６产生乳链菌肽（ｎｉｓｉｎ）的代谢调控及发酵条件进行研究，代谢动力学分析表明，ｎｉｓｉｎ在ＳＭ５２６指数生长期产生，到指数生长后期达到高峰，进入稳定期ｎｉｓｉｎ逐渐停止产生，表现出一种初级代谢动力学特征。ｎｉｓｉｎ的生物合成主要受碳源、氮源和磷源的调控，试验了各种不同的碳源，发现以二糖为碳源要比以单糖为碳源效果好；在基本发酵培养基中提高碳源（蔗糖）浓度，可使菌体得率和ｎｉｓｉｎ产率同时得以提高，其中以４％（Ｗ／Ｖ）蔗糖浓度较理想。此处碳源的调控主要是通过提高菌体生物量以达到提高ｎｉｓｉｎ效价。磷源则对ｎｉｓｉｎ的生物合成起极大的促进作用，试验了不同的磷源，其中以ＫＨ２ＰＯ４效果最好，在基本发酵培养基中提高ＫＨ２ＰＯ４浓度，可使得在菌体生物量基本不变的情况下，ｎｉｓｉｎ效价大幅度提高。