Polysaccharides can improve the survival of Lactiplantibacillus plantarum subjected to freeze-drying

Freeze-drying is one of the most commonly used methods of bacteria preservation. During this process, cryoprotectants can greatly reduce cellular damage. Micromolecular cryoprotectants have been widely adopted but have limited selectivity and protective effects. Therefore, explorations of other types of cryoprotectants are needed. This study aimed to explore the possibility of the macromolecular cryoprotectants and combinations of cryoprotectants to maintain bacterial activity. We found that the survival rate of Lactiplantibacillus plantarum AR113 after freeze-drying was 19% higher in the presence of soy polysaccharides than with trehalose, the best-performing micromolecular cryoprotectant. Moreover, a 90.52% survival rate of L. plantarum WCFS1 was achieved using the composite cryoprotectant containing soy polysaccharide and trehalose, which increased by 31.48 and 36.47% compared with adding solely trehalose or soy polysaccharide, respectively. These results demonstrate that macromolecular and micromolecular cryoprotectants have similar effects, and that combinations of macromolecular and micromolecular cryoprotectants have better protective effects. We further observed that the composite cryoprotectant can increase Lactobacilli survival by improving cell membrane integrity and lactate dehydrogenase activity. Our finding provides a new type of cryoprotectant that is safer and more effective, which can be extensively applied in the relevant food industry.     

Improvement of Freeze-Dried Lactobacillus Plantarum Survival Using Water Extracts and Crude Fibers from Food Crops

A synbiotic product of combined Lactobacillus plantarum TISTR 875 with water extracts and crude fibers from corn, mungbean, and soybean was formulated to investigate the survival of L. plantarum during freeze-drying and storage. The impacts of those water extracts and crude fibers on probiotic survival were determined in both a cultural medium and a freeze-drying medium. L. plantarum cultivated in de Man, Rogosa, and Sharpe (MRS) broth containing 2 % of water extract from soybean with 2 % mungbean fiber showed only 0.11 log CFU/ml cell reduction. The survival of L. plantarum harvested at the late log phase, mid stationary, phase and late stationary phase did not show statistical significance (P?>?0.05), whereas an initial pH of 6.5 and growth temperature of 37 °C showed greater impact (P?<?0.05). The addition of corn extract to the freeze-drying medium as a cryoprotectant had a similar effect on L. plantarum survival as sucrose, but it was better (P?<?0.05) than fructo-oligosaccharide and exopolysaccharides from Weissella cibaria A2, soybean extract, mungbean extract, soybean, corn, and mungbean fibers. A protective coating of corn extract was revealed and observed using scanning electron microscopy. The freeze-dried L. plantarum, cultivated in MRS broth containing soybean extract and mungbean fiber with corn extract as a cryoprotectant, retained high viability of 7.21 and 6.88 log CFU/ml after 8-week storage in a vacuum-packed aluminum foil-laminated polyethylene sachet and a nitrogen-flushed glass vial, respectively.     

Study of the internal mechanism of L-glutamate for improving the survival rate of Lactiplantibacillus plantarum LIP-1 after freeze-drying

Lactic acid bacteria are important fermentation strains, and improving their freeze-drying survival rate (FDSR) has become a hot research topic in recent years. In this research, we found that adding 0.1 g/L L-glutamate to culture medium improved the FDSR of four strains of L. plantarum. Using L. plantarum LIP-1 as an example, we explored the internal mechanism of this phenomenon. L-glutamate had this effect through two metabolic pathways. First, it made the strain grow rapidly and produce more acid, resulting in a rapid decline of pH in the culture environment. The acidic environment led to the upregulation of the dnaK gene and secG gene expression, increased DnaK protein levels, promoted its anchoring effect on the cell membrane, and enhanced the integrity of the cell membrane. Second, it upregulated the expression of the murL, murD, and vanY genes, increased peptidoglycan synthesis, and increased cell wall integrity. In sum, the strain obtained a higher FDSR by adjusting the cell membrane and cell wall structure using L-glutamate. This study provides a new method for improving the FDSR of lactic acid bacteria.     

嗜热链球菌M5-5冻干保护剂配方的优化

为了研究不同冻干保护剂在真空冷冻干燥过程中对嗜热链球菌M5-5菌体细胞的保护效果,采用单因素试验和正交试验,以冻干后的菌体存活率为评价指标,考察9种冻干保护剂在冷冻干燥过程中对M5-5菌体的保护效果,以期获得最优保护剂配方。结果表明,海藻糖、甘油和脱脂乳作为单一冻干保护剂时,菌体细胞存活率分别为43.85%、47.51%和40.86%,显著高于其他6种保护剂（P＜0.05）;通过正交试验确定冻干保护剂的最优配方为海藻糖60 g/L、谷氨酸钠20 g/L、甘油20 g/L、脱脂乳150 g/L,此时嗜热链球菌M5-5的冻干存活率可达88.41%。扫描电镜结果显示,添加保护剂配方组菌体细胞完整,表面光滑,说明该保护剂配方能有效减少冷冻干燥过程对菌体细胞的损伤。     

hs-3、ws-3乳酸菌发酵剂冻干研究

通过研究冻干保护剂的冻干厚度、干燥时间与冻干发酵剂含水量的关系,确定hs-3、ws-3乳酸菌冷冻干燥时间为13h,冻干厚度为0.75cm。对不同复合冷冻保护剂进行筛选,确定10%胡萝卜汁 6%葡萄糖 1%琼脂 1%麦芽糊精 1%番茄汁为乳酸菌优良的冷冻复合保护剂。冻干混合菌种发酵剂经发酵活力检测性能优良。     

应用新型复合保护剂制备高活性直投式干酪乳杆菌发酵剂

为应对直投式发酵剂在冷冻干燥处理中较易失活的问题,作者以干酪乳杆菌Lactobacilluscasei Zhang为研究对象,考察了不同冷冻干燥保护剂对菌体细胞生理活性的影响。研究表明,在以脱脂奶粉、山梨醇等传统冷冻干燥保护剂为保护基质的基础上,通过添加5 g/L谷胱甘肽能够将L.casei Zhang细胞的冻干存活率提高至54.5%,活菌数可达6.16×108cfu/mL。进一步分析表明,GSH使冷冻干燥后细胞的膜脂肪酸不饱和度(即U/S值)提高2.02倍,而饱和脂肪酸链长则从对照样本的17.1减少到16.0。此外,透射电镜的研究结果进一步证实,与对照样本相比,GSH能够有效维持冷冻干燥过程中细胞膜结构的完整性。上述研究结果对基于微生物细胞在冷冻干燥过程中的生理状态变化,开拓新型高效的冻干保护剂开辟了新的思路。     

Inactivation of Lactobacillus plantarum WCFS1 during spray drying and storage assessed with complementary viability determination methods

Survival of Lactobacillus plantarum WCFS1 spray-dried and stored under different conditions was investigated using complementary methods. One method involved a cell membrane integrity viability-based determination, the other assessed cell growth behavior in a liquid medium by means of detection time or by conventional plating. Survival decreased below 95% when spray drying was carried out at higher outlet spray drying temperatures (T_out > 70 °C). However, the membrane integrity method provided higher residual viability values compared to the detection time and conventional plating. This suggests that loss of viability may be due to a combination of damage to intracellular components and cell membrane. Also during storage viability based on growth behavior declined faster and was more temperature dependent compared to the viability as determined by the membrane integrity method. Also here additional damage to intracellular components is expected responsible to loss of viability. Major conclusion is that one should not only rely on a cell-membrane integrity based method to assess survival during spray drying and storage of bacteria. Previous studies that did so most probably underestimated viability as critical damage to intracellular components was not assessed.     

冻干乳酸双歧杆菌微胶囊的制备及存活性能研究

为了提高双歧杆菌在贮存和消费过程中的菌体存活率,利用冷冻干燥与微囊化结合的方法,将双歧杆菌和高效活菌保护剂一起作为核心物质,采用乳化法制备双歧杆菌微胶囊。通过正交试验,对双歧杆菌冻干过程中加入的保护剂种类及其最佳配比进行了研究。并探讨了双歧杆菌在模拟胃肠道环境以及高渗透压等极端环境条件下的存活能力。结果表明：加入10%菊糖、6%大豆蛋白、12%海藻糖、10%甘露醇可显著提高微囊化双歧杆菌冻干活菌数,使其存活率达到69.2%。较之冻干菌粉,双歧杆菌微囊在模拟胃液、肠液、高胆汁盐以及高渗透压溶液中均具有较好的耐受性。     

Probiotic potential of Lactobacillus strains with anti-allergic effects from kimchi for yogurt starters

Lactobacillus strains were isolated from kimchi and investigated for their potential use for probiotic yogurt starters. Two of the isolated strains have over 90% survival rate to artificial gastric acid (pH 2.5, 1% pepsin) and artificial bile acid (0.3% oxgall). These strains were identified as Lactobacillus plantarum by 16S rRNA sequencing, and named as L. plantarum SY11 and L. plantarum SY12. The known carcinogenic enzyme, β-glucuronidase was not produced by L. plantarum SY11 and L. plantarum SY12. These 2 strains were found to be resistant to several antibiotics and strongly adhered to intestinal cells. Antiallergic effect of L. plantarum SY11 and L. plantarum SY12 was demonstrated using nitric oxide (NO) and cytokine production. L. plantarum SY11 and L. plantarum SY12 were capable of significantly decreasing NO production, and reduced T helper 2-associated cytokines, cyclooxygenase-2, tumor necrosis factor-α, and inducible nitric oxide synthase compared to control. Yogurt samples produced using L. plantarum SY11 and L. plantarum SY12 did not show significant differences in microbiological, physicochemical, and sensory properties compared with yogurt sample produced using commercial strain. Therefore, these two strains could be used as probiotic yogurt starters with antiallergic effects.     

黑曲霉孢子粉真空冷冻干燥保护剂配方优化

采用单因素试验和混合均匀试验设计,对黑曲霉孢子粉真空冷冻干燥保护剂配方进行筛选和优化。通过Design-Expert软件对实验数据进行回归分析,得到保护剂最佳配方为脱脂奶粉6.43 g/100 mL、蔗糖13.03 g/100 mL、甘油6.88 g/100 mL,孢子存活率预测值为88.82%,实际值为87.03%。     

响应面法优化乳酸片球菌冻干保护剂配方

添加冻干保护剂能有效提高微生物冻干存活率。为了提高乳酸片球菌冻干存活率,采用Plackett Burman实验设计与最陡爬坡实验结合响应面法,研究了不同冻干保护剂对冻干存活率的影响。结果表明,海藻糖、甘露醇、硫酸锰浓度对菌体存活率影响显著,海藻糖与甘露醇交互作用显著,海藻糖与硫酸锰,甘露醇与硫酸锰交互作用不显著。最佳保护剂配方:海藻糖浓度为73.64 g/L,甘露醇浓度为43.76 g/L,硫酸锰浓度为1.91 g/L,冻干存活率为98.10%±1.03%。实验结果为益生菌生产提供了技术支持。     

冷冻保护剂对直投式木葡萄酸醋杆菌发酵剂菌体细胞特性的影响

本文研究了1%谷氨酸、5%海藻糖、10%脱脂乳及复合0.575%谷氨酸+0.075%海藻糖+6.4%脱脂乳作为冷冻保护剂对直投式木葡萄酸醋杆菌(GBX)发酵剂菌体细胞特性的影响,通过测定GBX发酵剂菌体细胞的DNA泄露、细胞液总抗氧化能力、SOD活性、纤维素合成酶活性和ATP酶活性变化等情况来衡量GBX菌体细胞的生理活性变化。结果发现:添加单一1%谷氨酸、5%海藻糖、10%脱脂乳以及复合0.575%谷氨酸+0.075%海藻糖+6.4%脱脂乳作为保护剂均能明显抑制冷冻干燥过程中菌体细胞的DNA泄露,提高细胞总抗氧化能力、SOD活性、纤维素合成酶和ATP酶活性,表明添加冷冻保护剂能够一定程度上保护菌体细胞,其中复合保护剂对菌体细胞的保护效果最佳,电子扫描显微镜也验证了冷冻保护剂能确实降低菌体细胞在冷冻干燥过程中损害程度,从而使菌体细胞保持良好的生理活性。     

Pressure-resistant acclimation of lactic acid bacteria from a natural fermentation product using high pressure

Twenty colonies of Pediococcus acidilactici, Lactobacillus brevis, Lactiplantibacillus plantarum and Latilactobacillus curvatus were isolated from fermentation broth of pickled pepper by using MRS agar. The strains were acclimated using high pressure from 300 to 400 MPa for 1 to 5 min. For one generation of acclimation, the survived cells after first high pressure treatment were then activated by culturing on MRS agar for two days for the next high pressure treatment. After acclimation for 30 generations, the survival ratio of L. plantarum and L. curvatus was increased by 138 and 1222 times at 400 MPa for 1 and 3 min, respectively. Weibull model could appropriately fit the survival curves of L. plantarum and L. curvatus treated by high pressure. The time to the first decimal reduction of these two strains after acclimation was 3.5 and 1.8 times, respectively, longer than before acclimation at 300 MPa. From electron microscopy photographs, the acclimated L. curvatus was more curved, smaller and its cell wall was thicker than the unacclimated L. curvatus. After HPP treatment, L. plantarum and L. curvatus have higher cell integrity, smoother cell surface, more uniform protoplasm and smaller cavities in the cell protoplasm compared with unacclimated strains, suggesting that high pressure acclimation introduced some modification to bacteria intrinsic structures and increased their pressure resistance. Moreover, the fermentation performance including glucose utilization capacity and lactic acid production of the acclimated L. curvatus was respectively improved by 9.6% and 9.4% at 37 °C for 24 h in MRS medium.     

Microbial association and acidity development of unheated and pasteurized green-table olives fermented using glucose or sucrose supplements at various levels

Green-table olives, unheated and pasteurized, of Greek cultivar Conservolea were supplemented with glucose or sucrose in various amounts inoculated with Lactobacillus plantarum and fermentation was conducted. Although sugar supplements did not affect the lactic acid bacteria growth rate, they increased the rate of pH drop and the production of acids and lowered the final pH values in all cases. Adequate supplements of sugars (0.5%, 1.0% w v⁻¹), of both types, in unheated olive fermentation, resulted in a fast pH drop, causing a satisfactory halt of Enterobacteriaceae growth in the first days of fermentation, and a subsequent population decline occurred in the following days, eliminating the danger of early stage spoilage and ensuring the safety of the final product. In pasteurized olives, where L. plantarum starter was the only microbial flora, it was considered worth evaluating the fermentation progress without any competition. The rate of pH drop was not as high as in unheated olives, but final pH values and acid development were more pronounced. Lactic acid was the predominant acid developed in both unheated and pasteurized olive fermentation, and increased sugar supplements resulted in faster production and higher yields of this acid. Acetic acid was also produced in low amounts at the end of fermentation, except in the cases of pasteurized olive fermentation supplemented with sucrose, where acetic acid was absent. A switch from homo-fermentative to hetero-fermentative metabolism might have taken place in case of glucose presence, but the same was not observed for sucrose.     

Cellular injuries of spray-dried Lactobacillus spp. isolated from kefir and their impact on probiotic properties

The injuries caused by spray drying (SD) of three potential probiotic lactobacilli isolated from kefir grains and the impact on some probiotic properties, were evaluated. Results demonstrated that Lactobacillus plantarum 83114 and L. kefir 8321 showed a slight reduction of viability (0.11 and 0.29 log CFU/ml respectively) after SD process, and L. kefir 8348 was found to be more sensitive to the process with a reduction in viability of 0.70 log CFU/ml. Neither membrane damage, evaluated by increased sensitivity to NaCl, lysozyme, bile salt and penicillin G, nor changes in acidifying activity in MRS and milk by lactobacilli were detected after SD. L. plantarum 83114 and L. kefir 8321 after SD did not lose their capacity to adhere to intestinal cells. Nevertheless, L. kefir 8348 showed a significant loss of adhesion capacity after SD. In addition, rehydrated spray-dried L. kefir 8321 retained the ability to protect against Salmonella invasion of intestinal cells. This effect was observed when L. kefir is co-incubated with Salmonella before invasion assay.This work shows that the membrane integrity evaluated by indirect methods and some probiotic properties of lactobacilli isolated from kefir did not change significantly after SD, and these powders could be used in functional foods applications.     

鲢鱼冷冻鱼糜抗冻剂的复配研究

选取功能性食品抗冻剂海藻糖和乳糖醇,并与传统商业抗冻剂混合,以得到“低甜、低热”且经济适用的复合抗冻剂。以白度、保水性、凝胶强度和盐溶性蛋白为指标,通过L9(34)正交试验探寻新的冷冻鱼糜复合抗冻剂配方。结果表明最佳配方为：海藻糖4%、乳糖醇3%、蔗糖1%、山梨醇2%,新的复合抗冻剂比商业抗冻剂甜度低,且能达到理想的抗冻效果。     

Effects of different drying treatments on the stability of carotenoids in Taiwanese mango (Mangifera indica L.)

The stability of carotenoids in Taiwanese mango as affected by different drying treatments was studied. Mangoes were soaked in 1% sodium hydrogen sulfite solution or 1% ascorbic acid solution, prior to hot-air drying and freeze-drying. Results showed that in most cases, the highest yield of the epoxy-containing carotenoids was achieved by freeze-drying plus soaking in 1% sodium hydrogen sulfite solution. However, freeze-drying plus soaking in 1% ascorbic acid solution resulted in the highest retention of all-trans-β-carotene and its cis isomers, all-trans-zeaxanthin and its cis isomers, as well as cis-lutein. Nevertheless, for hot-air drying, with or without soaking, a mango product of deep orange colour was produced. On freeze-drying, mango could generate yellow colour, while a lighter color was observed when soaked in antioxidants.     

Lactobacillus plantarum—survival,functional and potential probiotic properties in the human intestinal tract

Lactobacillus plantarum is a versatile lactic acid bacterium that is encountered in a range of environmental niches, has a proven ability to survive gastric transit, and can colonize the intestinal tract of human and other mammals. Several studies describe the effects of L. plantarum consumption on human physiology. The availability of the complete genome sequence of L. plantarum WCFS1 makes it a suitable model to explore the molecular mechanisms underlying the targeted intestinal properties of this species. An increasing number of studies address the development of L. plantarum into an ingestible living vaccine. Furthermore, studies are emerging to determine the activity of L. plantarum in the human intestinal tract. This review discusses the studies of the safety and survival of L. plantarum in the human intestinal tract, the effects of this bacterium on the host and it provides an overview of the molecular studies addressing the activity of L. plantarum in the human gut environment.     

Cryoprotectant Effects during Short Time Frozen Storage of Chicken Myofibrillar Protein Isolates

The purpose of our study was to determine whether stabilization of myofibrillar protein isolates (MPI) during frozen storage could be achieved by addition of certain cryoprotectants. For 2–4 wk at - 21°C MPI were exposed to different freezing and thawing treatments to also determine to what extent cryoprotectants may prevent denaturation of MPI. Overall evaluation of color, weight losses of gels during cooking as well as texture proved that 2.8% sorbitol in combination with 4% starch was the best cryoprotectant. A positive effect was also noted for a mixture of 2.8% sorbitol and 4% sucrose. In contrast, the addition of a dextrose polymer mixture to MPI was not effective in maintaining product integrity.     

酸-冷交互胁迫对保护冷冻干燥发酵乳杆菌活性的作用

为探究酸-冷交互胁迫对冷冻干燥发酵乳杆菌(Lactobacillus fermentum)ATm的影响,考察酸-冷交互胁迫的处理方式对细胞存活率、滞后时间以及酸化速率的影响,同时测定交互胁迫对乳酸脱氢酶、ATP酶活性以及细胞膜完整性的影响。结果表明,pH 4.5、4℃酸-冷交互胁迫能够显著提高细胞冷冻干燥存活率及酸化速率,其中冷冻干燥存活率能够达到87.19%,优于单因素胁迫;pH 4.5、4℃酸-冷交互胁迫能够提高发酵乳杆菌ATm的冷冻干燥存活率保护其活性,提高酶活性,维持细胞膜完整性。