Enhancement of functional characteristics of mixed lactic culture producing nisin z and exopolysaccharides during continuous prefermentation of milk with immobilized cells

Antagonistic phenomena between strains often occur in mixed cultures containing a bacteriocinogenic strain. A nisin Z producer (Lactococcus lactis ssp. lactis biovar. diacetylactis UL719) and 2 nisin-sensitive strains for acidification (Lactococcus lactis ssp. cremoris ATCC19257) and exopolysaccharide (EPS) production (Lactobacillus rhamnosus RW-9595M) were immobilized separately in gel beads and used to continuously preferment milk at different temperatures, with pH controlled at 6.0 by fresh milk addition. The process showed high volumetric productivity, with an increase from 8.0 to 12.5 L of prefermented milk per liter of reactor volume and hour as the temperature was increased from 27 to 35°C. Lactococcus lactis ssp. lactis biovar. diacetylactis UL719 counts in prefermented and fermented (22-h batch fermentation) milks were stable during 3 wk of continuous fermentation (8.1 ± 0.1 and 8.9 ± 0.2 log cfu/mL, respectively). The L. lactis ssp. cremoris population (estimated with real-time quantitative PCR) decreased rapidly during the first week of continuous culture to approximately 4.5 log cfu/mL and remained constant afterward. Lactobacillus rhamnosus counts in prefermented and fermented milks significantly increased with prefermentation time, with no temperature effect. Nisin Z reached high titers in fermented milks (from 177 to 363 IU/mL), with EPS concentration in the range from 43 to 178 mg/L. Immobilization and continuous culture led to important physiological changes, with Lb. rhamnosus becoming much more tolerant to nisin Z, and Lb. rhamnosus and L. lactis ssp. lactis biovar. diacetylactis UL719 exhibiting large increases in milk acidification capacity. Our data showed that continuous milk prefermentation with immobilized cells can stimulate the acidification activity of low-acidifying strains and produce fermented milks with improved and controlled functional properties.     

以乳清为原料采用混菌发酵技术促进Nisin生产的研究

采用了一种不用外加碱或移除乳酸来达到控制发酵液中乳酸的新技术,即采用乳酸菌和酵母菌的混和发酵来控制pH值以促进乳酸链球菌素(nisin)的生产。其原理是乳酸菌在发酵生产nisin的过程中所产生的乳酸可以被酵母菌利用,从而达到控制发酵液pH值的目的;同时,混菌培养选用的酵母菌不能利用乳清培养基中的乳糖,从而避免了2株菌之间对碳源的竞争,创造了一个互利共生的微生态环境,从而达到促进nisin生产的目的。实验表明,乳酸根的积累对nisin发酵生产具有抑制作用;混合发酵的酵母菌利用了发酵液中的乳酸,促进了乳酸菌生长和nisin生产;酵母菌较乳酸菌提前3h接入到发酵培养基中时,能更好地控制pH值;最佳的接种比例为乳酸菌3%,酵母菌5%。     

Impact of nisin producing culture and liposome-encapsulated nisin on ripening of Lactobacillus added-Cheddar cheese

This study aimed to evaluate the effects of incorporating liposome-encapsulated nisin Z, nisin Z producing Lactococcus lactis ssp. lactis biovar. diacetylactis UL719, or Lactobacillus casei-casei L2A adjunct culture into cheese milk on textural, physicochemical and sensory attributes during ripening of Cheddar cheese. For this purpose, cheeses were made using a selected nisin tolerant cheese starter culture. Proteolysis, free fatty acid production, rheological parameters and hydrophilic/hydrophobic peptides evolution were monitored over 6 mo ripening. Sensory quality of cheeses was evaluated after 6 mo. Incorporating the nisin-producing strain into cheese starter culture increased proteolysis and lipolysis but did not significantly affect cheese rheology. Liposome-encapsulated nisin did not appear to affect cheese proteolysis, rheology and sensory characteristics. The nisinogenic strain increased the formation of both hydrophilic and hydrophobic peptides present in the cheese water extract. Sensory assessment indicated that acidic and bitter tastes were enhanced in the nisinogenic strain-containing cheese compared to control cheese. Incorporating Lb. casei and the nisinogenic culture into cheese produced a debittering effect and improved cheese flavor quality. Cheeses with added Lb. casei and liposome-encapsulated nisin Z exhibited the highest flavor intensity and were ranked first for sensory characteristics.     

Inhibition of Clostridium tyrobutyricum in Vidiago cheese by Lactococcus lactis ssp. lactis IPLA 729, a nisin Z producer

Lactococcus lactis ssp. lactis IPLA 729 is a nisin Z producer isolated from raw milk cheese able to grow and produce nisin Z in milk. The ability of this strain to inhibit the growth of Clostridium tyrobutyricum CECT 4011, a late blowing agent, in Vidiago cheese, a semi-hard farmhouse variety, manufactured in Asturias, Northern Spain, was investigated. For control purposes, cheeses were manufactured with the mesophilic mixed starter IPLA-001. In experimental cheeses, the nisin-producing strain L. lactis IPLA 729 was combined with this starter. Nisin Z activity reached a concentration of 1600 AU/ml in 1-day cheeses and this level was maintained until 15 days of ripening. Furthermore, to compare the inhibitory activity of the nisin-producing strain to nitrate, cheeses were also manufactured with a commercial starter culture and potassium nitrate as anti-blowing agent was added in accordance with Vidiago's cheesemakers. The control, experimental and commercial cheeses were contaminated with C. tyrobutyricum CECT 4011. The composition of the three different cheeses showed only slight differences with respect to total solids, protein and fat, although control and experimental cheeses showed a richer flavour-compound profile than commercial cheeses. The level of the spoilage strain C. tyrobutyricum CECT 4011 decreased from 1.2x10(6) to 1.3x10(3) cfu/g during ripening in presence of the nisin Z producer, while it increased to 1.99x10(9) cfu/g in control cheeses and to 3.5x10(7) cfu/g in commercial cheeses.     

荞麦鲜奶酪发酵工艺研究及其质量评价

以牛乳、荞麦为原料,开发研制荞麦鲜奶酪。在单因素试验的基础上,采用响应面分析方法,优化荞麦添加量、发酵温度与发酵时间的工艺参数,并对最终产品进行质量评价。荞麦鲜奶酪生产的最优工艺参数为荞麦添加量4%、发酵剂添加量0.015%、发酵温度42.6℃、发酵时间7.1 h。所制备的酸乳凝乳状态良好,乳清析出少且澄清,荞麦鲜奶酪咀嚼性适中,带有荞麦独有的香气,且质量较优,是一种营养丰富的特色鲜奶酪。     

Liposome encapsulated nisin Z: optimization,stability and release during milk fermentation

Liposomes prepared from different proliposomes (Pro-lipo^® H, Pro-lipo^® S, Pro-lipo^® C and Pro-lipo^® DUO) were tested for their capacity to encapsulate nisin Z. Factors affecting the entrapment process (pH and concentration of nisin Z solution and cholesterol concentration in the lipid membranes) were optimized. The use of nisin Z monoclonal antibodies made it possible to quantify, using a competitive enzyme immunoassay, and visualize, using transmission electron microscopy, nisin Z. Nisin Z was entrapped in different liposomes with encapsulation efficiencies (EE) ranging from 9.5% to 47%. The pH of nisin Z aqueous solution and nisin Z concentration had a significant effect on the amount of encapsulated nisin. An increase in cholesterol content in lipid membranes up to (20%, w/w) resulted in a slight reduction in EE. Nisin-loaded vesicles did not severely disturb Cheddar cheese fermentation and showed stability to Cheddar cheese temperature cycle. Long term stability of liposome-encapsulated nisin Z was demonstrated for 27 days at 4°C in different media including milk with different fat levels (3.25%, 2.0% and 1.0%), skim milk, sweet whey and phosphate buffer saline (PBS). Liposome stability determined as the quantity of released nisin Z was highest in milk followed by PBS and whey, respectively.     

Efficacy of bovicin HC5 and nisin combination against Listeria monocytogenes and Staphylococcus aureus in fresh cheese

Fresh and soft cheeses provide a suitable medium for the growth of many microorganisms and have been frequently associated with foodborne diseases. This study aimed to evaluate the effects of the bacteriocins bovicin HC5 and nisin on Listeria monocytogenes and Staphylococcus aureus in Minas Frescal cheese, a traditional Brazilian fresh product. The cheese was inoculated with 10⁴ CFU g^?1 of both pathogens, and the bacteriocins were added at 600 AU g^?1 of each one. After 9 days of storage at 4 °C, L. monocytogenes Scott A were not detected in 25 g samples. Although the bacteriocins reduced the population of S. aureus ATCC 6538, increasing numbers were detected after 15 days of storage at 4 °C. Staphylococcal enterotoxin C was detected in cheeses added of bacteriocins and stored under abusive temperature of 15 °C. The results demonstrate the potential application of combination of bovicin HC5 and nisin in controlling the pathogens growth assessed in Minas Frescal cheese.     

Effects of mixed starter composition on nisin Z production by lactococcus lactis subsp. lactis biovar. diacetylactis UL 719 during production and ripening of Gouda cheese

A starter culture system that produced both acid and nisin at acceptable rates in milk for manufacture of Gouda cheese was developed using nisin Z-producing L. lactis subsp. lactis biovar. diacetylactis UL 719 (UL 719) and a commercial Flora Danica (FD) starter culture. Different compositions of mixed cultures (0, 0.2, 0.4, 0.6 or 0.8% UL 719 with 1.4% FD) were tested for acidification and nisin Z production in milk after 12 h incubation at 30 degrees C. The 0.6/1.4% combination, selected as the optimal mixture of starter cultures, acidified milk to a suitable pH and produced nisin Z at a high concentration of 512 IU/ml. With this optimal combination, FD numbers of citrate-fermenting and non-fermenting bacteria did not change compared with the control (1.4% FD). However, with 0.8% of L. lactis strain UL 719 and 1.4% of the FD starter culture, the numbers of citrate-fermenting and non-fermenting bacteria in fermented milk decreased compared with those obtained when milk was inoculated with 0.2, 0.4 or 0.6% of UL 719 added to 1.4% FD or control cultures (1.4% FD). Mixed starter culture ratios 0.6/1.4%, 0.4/1.4% and 0.5/1.4% (UL 719/FD) were used to manufacture nisin Z containing Gouda cheese which was ripened up to 45 weeks. The composition of control cheeses made with 1.4% FD, and nisin Z-containing Gouda cheeses were similar with respect to percent moisture, fat, salt and protein. During the ripening period, the cell counts observed were approximately two logs higher in cheese made with the 0.6/1.4% mixed starter culture than in control cheese. In experimental cheese produced with 0.6/1.4% (UL 719/FD) mixed starter culture, nisin activity increased from 256 IU/g at the end of manufacture to a maximum of 512 IU/g after 6 weeks of ripening; the levels then decreased to 128 and 32 IU/g after 27 and 45 weeks of ripening, respectively. In contrast, nisin Z was not detected in experimental cheeses made with 0.4/1.4% or 0.5/1.4% (UL 719/FD) mixed starters. Using an affinity purified anti-nisin polyclonal antibody, anti-rabbit gold-conjugate and transmission electron microscopy, nisin Z was found to be localized in the cheese matrix, in fat globules, in the casein phase and concentrated at the fat-casein interface. After 27 weeks of ripening, nisin Z was detected preferentially in the fat globules of the experimental cheese.     

混合发酵生产功能性饮料的开发与研究进展

目前市场上功能性饮料层出不穷,种类繁多,但没有统一的标准。文中从混合发酵角度出发,阐述了发酵生产功能性饮料的种类以及开发研究状况,还指出了混合发酵生产的共性和存在的一些问题,为科学研发提供参考依据。     

Occurrence of nisin Z production in Lactococcus lactis BFE 1500 isolated from wara, a traditional Nigerian cheese product

Screening for bacteriocin production of 500 strains of lactic acid bacteria (LAB) from various African fermented foods resulted in the detection of a bacteriocin producing Lactococcus lactis (BFE 1500) isolated from a dairy product called wara. The bacteriocin inhibited not only the closely related LAB, but also strains of Listeria monocytogenes, Listeria innocua, Clostridium butyricum, Clostridium perfringens, Bacillis cereus and Staphylococcus aureus. It was heat stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10), but highest activity was observed in the lower pH range. The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K, but not by other proteases. Growth kinetic assay indicated stronger growth inhibition by the bacteriocin produced by Lc. lactis BFE 1500 on L. monocytogenes WS 2250 and B. cereus DSM 2301 than with the nisin A producing strain DSM 20729. Polymerase chain reaction indicated the presence of the nisin operon in strain BFE 1500 and sequencing of its structural gene showed that Lc. lactis BFE 1500 produced the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The genetic determinants for bacteriocin production in strain BFE 1500 are located on a conjugative transposon. The ability of the bacteriocin produced by Lc. lactis BFE 1500 to inhibit a wide range of food-borne pathogens is of special interest for food safety, especially in the African environment with perennial problems of poor food hygiene.     

混菌固态发酵生产菜籽肽培养基条件优化研究

以工业副产品菜籽粕为原料,通过枯草芽孢杆菌和雅致放射毛霉混菌固态发酵生产菜籽肽。先以肽得率、氮溶解指数和硫甙降解率为指标通过单因素实验初步得到混菌发酵的培养基条件,再根据Box-Benhnken的中心组合实验设计原理,在单因素实验的基础上采用响应面分析法,建立起菜籽肽得率与各影响因素的回归方程,优化混菌固态发酵培养基组成,得出最佳的培养基工艺条件为:菜籽粕中麸皮添加量5%、料液比1:1.35、葡萄糖添加量0.50%、KH2PO4添加量0.36%、初始pH6.5,此条件下发酵产品的菜籽肽得率可达6.85%,同时测得在此优化条件下的硫甙降解率为62.09%。     

混菌固态发酵生产蓖麻饼肥工艺条件的优化

采用产纤维素酶菌株T1-2和产蛋白酶菌株P3-2进行固态发酵蓖麻饼粕,降解其中的大分子物质。通过单因素试验,以还原糖和游离氨基酸含量为评价指标,得到最佳发酵条件为:水分含量40%,发酵时间5d,菌种比例为1∶1,接种量14%,发酵温度30℃,pH值为4,麸皮含量20%。以游离氨基酸含量为评价指标,经正交试验得优化培养条件为培养基含水量50%、接种量15%、36℃、发酵7d,在此条件下发酵后游离氨基酸含量达1.68%。     

多菌种混合发酵黑米饮料的研究

研究了以黑米为原料,通过蒸煮、糖化制得黑米汁,再接入酵母菌、乳酸菌、醋酸菌共同发酵所得的一种新型发酵无醇饮料。试验表明,黑米汁发酵的最佳糖度为12°Bx,pH为6.5,接入菌种的比例为酵母菌∶乳酸菌∶醋酸菌=0.5∶1∶2,接种量为0.1g/L,发酵时间为8～9d,发酵温度为33℃。所得发酵液经调配、杀菌便制成了黑米发酵无醇饮料。     

Analysis of nisin A,nisin Z and their degradation products by LCMS/MS

The peptides nisin A and nisin Z belong to type-A lantibiotics applied as preservatives in cheese production. The present study optimised and validated a liquid chromatography–tandem mass spectrometry (LCMS/MS) method for the analysis of nisin A in cheese. Since nisin A was not detectable in nisin-containing commercial cheese samples, an additional LCMS/MS method for the quantification of nisin Z was developed and validated. Quantification was performed by external calibration and standard addition. The latter method provided a non-significantly higher recovery rate for the tested cheese matrix. During the production of processed cheese, nisin A and nisin Z undergo significant degradation. Six degradation products of nisin A or nisin Z, respectively, were detected and assigned to nisin A/Z + H₂O, nisin A/Z^1–32, and nisin A/Z^1–32 + H₂O. In two out of eight commercial processed cheese samples, 1.6, resp. 1.7 mg nisin Z/kg cheese was measured, whereas nisin A was not detectable in any of the samples.     

Detoxification of cassava during fermentation with a mixed culture inoculum

The efficacy of a mixed culture inoculum in detoxifying intact cassava tuber and peel pieces was investigated. Fermented cassava tuber and peel pieces had more linamarase activity than the non-fermented control samples and this might have resulted partly from the release of endogenous linamarase from the retted tuber/peel pieces and partly due to the microbial linamarase. Approximately 24–26% of the total cyanide remained in the bound form in the fermented tuber while 67–79% existed as bound cyanide in the non-fermented controls. In cassava peels, only 15–33% of total cyanide remained in bound form after 72 h fermentation while 58–59% cyanide existed in bound form in the parallel non-fermented control peels. Free cyanide which could be easily eliminated through sun-drying was present at higher concentrations in the fermented tuber and peel compared with the non-fermented samples. The study showed that cassava tuber and peel could be extensively detoxified through fermentation with a mixed culture inoculum.     

红曲霉液态发酵生产红色素的培养基配方研究

研究了菌株紫红曲霉No.1-18-54液态发酵产红色素的培养基配方,通过对比试验和正交试验,其最佳液体培养基配方为大米粉9%、硝酸钠0.2%、磷酸二氢钾0.1%、硫酸镁0.2%。     

维生素C二步发酵菌发酵条件的优化

目的:改变发酵条件以提高发酵收率.方法:优化pH值、通风量、微量元素等发酵因子,找到最佳使用量.结果:发酵培养基中L-山梨糖起始浓度8%、碳酸钙浓度0.05%、复合VB以及分段调节pH值、通气量等可以有效地提高2-酮基-L-古龙酸的产量.结论:通过优化发酵条件可以优化发酵菌系的生理状态,促进大小菌之间的协调,使其达到最佳发酵状态.     

产雌马酚菌与肠杆菌混合发酵的研究

雌马酚是大豆异黄酮在结肠中特定微生物作用下的降解产物,具有多种生物学活性。从婴儿肠道中筛选到一株可以降解大豆异黄酮产生雌马酚的厌氧菌株粪链球菌BY-1,研究了肠杆菌BY-2对BY-1的生长和代谢的影响,并对其相互作用机理进行了初步探讨。结果表明,BY-1与BY-2以1:1的接种比例混合培养时,BY-1的产酸量和雌马酚产量都显著高于单独培养时。肠杆菌BY-2代谢产生的酶、有机酸、氢气都可能是促进粪链球菌BY-1生长的因素,BY-1与BY-2之间是偏利关系。为开发含雌马酚保健发酵乳提供了一定的参考价值。     

混合菌种固态发酵大豆粕生产大豆肽研究

大豆肽系由大豆蛋白经水解所得由3～6个氨基酸残基组成低分子肽混合物,分子量以低于1000Da为主。以大豆粕为原料,采用黑曲霉、米曲霉混合菌种固态发酵法生产大豆肽,所得大豆肽具有较好理化特性和生理活性,克服酶解法产品苦味重、口感差等缺点,可在很多领域得以广泛应用。     

Selection and identification of protective culture for controlling Staphylococcus aureus in fresh Domiati like cheese

Antibacterial activity of forty lactic acid bacteria (LAB) isolates toward Staphylococcus aureus was evaluated. The selected strains were then used as protective culture in artificial contaminated Domiati like cheese with S. aureus. The effect of using these strains on physicochemical properties and overall acceptability of fresh cheese was evaluated. Depending on its antibacterial activity, three strains of Lactobacillus rhamnosus 130RZFAAU, 131RZFAAU, and 190RZFAAU were selected for cheese making. No negative sensory properties were observed by the panelists when LAB strains were used as a single culture in the fresh cheese making. The application of these strains as protective culture in artificial contaminated cheesemaking process give a positive results. S. aureus was detected in cheese samples by culture method and propidium mono azide–quantitative polymerase chain reaction method. The results recommended that the strain L. rahmnosus 131RZFAUU that used in this study has antimicrobial activity against S. aureus and could be used as protective culture for improving the safety of Egyptian soft cheese.

Practical applications

Detection of pathogenic bacteria by classical tests can take several days. It would be useful to have a rapid detection protocol to screen for the presence of Staphylococcus aureus in milk and cheese. Application of real‐time PCR in cheese is sufficient in characterization the S. aureus communities in raw milk and follow the dynamics of the entire populations in cheese. Recently, some scientific publications have shown that the naturally cheese microflora can efficiently prevent the growth of pathogenic or spoilage microorganisms. The control of spoilage and pathogens bacteria has been traditionally done by chemical additives, but the application of promising protective cultures, especially for traditionally cheeses made from raw milk, is limited. This work present some protective culture selected for controlling S. aureus in soft cheese. This work confirm the PMA‐q PCR method for detection live cells of S. aureus in cheese rapidly.