Freeze-dried Lactic Acid Bacteria Research Articles

Supercooling Pretreatment Improves the Shelf-Life of Freeze-Dried Leuconostoc mesenteroides WiKim32.

Storage stability of freeze-dried lactic acid bacteria is a critical factor for their cost-effectiveness. Long-term storage of lactic acid bacteria enables microbial industry to reduce distribution costs. Herein, we investigated the effect of cold adaptation under supercooling conditions at -5°C on the viability of Leuconostoc mesenteroides WiKim32 during the freeze-drying process and subsequent storage. Cold adaptation increased the thickness of exopolysaccharides (EPS) and improved the viability of freeze-dried Leu. mesenteroides WiKim32. Compared to non-adapted cells, cold-adapted cells showed a 35.4% increase in EPS thickness under supercooling conditions. The viability of EPS-hydrolyzed cells was lower than that of untreated cells, implying that EPS plays a role in protection during the freeze-drying process. Cold adaptation increased the storage stability of freeze-dried Leu. mesenteroides WiKim32. Fifty-six days after storage, the highest viability (71.3%) was achieved with cold adaptation at -5°C. When EPS-containing broth was added prior to the freeze-drying process, the viability further increased to 82.7%. These results imply that cold adaptation by supercooling pretreatment would be a good strategy for the long-term storage of Leu. mesenteroides WiKim32.

Effects of sucrose, carnosine, and their mixture on the glass transition behavior and storage stability of freeze-dried lactic acid bacteria at various water activities

Effects of sucrose, carnosine, and their mixture on the glass transition behavior and storage stability of freeze-dried Lactobacillus reuteri at various water activities (aw) were investigated. At aw = 0.328, the control (non-additive sample) showed viable cells as uncountable after storage at 25 °C for 4 weeks. The sucrose and sucrose-carnosine samples showed clear glass transition at a slightly lower temperature than the storage temperature, and maintained a large number of viable cells after storage. The carnosine sample crystalized during the storage, and a large reduction in viable cells was observed. At aw = 0.576, the samples showed a small endothermic shift due to glass transition, suggesting partial crystallization. The Tg decreased with increase in aw because of the water plasticizing effect. After storage, the sucrose-carnosine sample showed much higher viable cell numbers than the other samples. At aw = 0.753, the sucrose and sucrose-carnosine samples showed clear glass transition. The carnosine sample showed freeze-concentrated glass transition and subsequent ice melting. After storage, the sucrose and carnosine samples showed an uncountable and a low number of viable cells, respectively, but sucrose-carnosine maintained relatively high viable cell numbers. In addition, carnosine strongly supported the stabilizing effect of sucrose (even at low additive levels) depending on the aw. These results suggest that sucrose-carnosine shows a synergistic stabilizing effect.

Effect of Protectants Made From Sucrose and Antioxidant Blends On The Stability of Freeze-Dried Lactic Acid Bacteria

Effect of Protectants Made From Sucrose and Antioxidant Blends On The Stability of Freeze-Dried Lactic Acid Bacteria

Design of a new lyoprotectant increasing freeze-dried Lactobacillus strain survival to long-term storage

BackgroundStabilization of freeze-dried lactic acid bacteria during long-term storage is challenging for the food industry. Water activity of the lyophilizates is clearly related to the water availability and maintaining a low aw during storage allows to increase bacteria viability. The aim of this study was to achieve a low water activity after freeze-drying and subsequently during long-term storage through the design of a lyoprotectant. Indeed, for the same water content as sucrose (commonly used lyoprotectant), water activity is lower for some components such as whey, micellar casein or inulin. We hypothesized that the addition of these components in a lyoprotectant, with a higher bound water content than sucrose would improve lactobacilli strains survival to long-term storage. Therefore, in this study, 5% whey (w/v), 5% micellar casein (w/v) or 5% inulin (w/v) were added to a 5% sucrose solution (w/v) and compared with a lyoprotectant only composed of 5% sucrose (w/v). Protective effect of the four lyoprotectants was assessed measuring Lactiplantibacillus plantarum CNCM I-4459 survival and water activity after freeze-drying and during 9 months storage at 25 °C.ResultsThe addition whey and inulin were not effective in increasing Lactiplantibacillus plantarum CNCM I-4459 survival to long-term-storage (4 log reduction at 9 months storage). However, the addition of micellar casein to sucrose increased drastically the protective effect of the lyoprotectant (3.6 log i.e. 0.4 log reduction at 9 months storage). Comparing to a lyoprotectant containing whey or inulin, a lyoprotectant containing micellar casein resulted in a lower water activity after freeze-drying and its maintenance during storage (0.13 ± 0.05).ConclusionsThe addition of micellar casein to a sucrose solution, contrary to the addition of whey and inulin, resulted in a higher bacterial viability to long-term storage. Indeed, for the same water content as the others lyoprotectants, a significant lower water activity was obtained with micellar casein during storage. Probably due to high bound water content of micellar casein, less water could be available for chemical degradation reactions, responsible for bacterial damages during long-term storage. Therefore, the addition of this component to a sucrose solution could be an effective strategy for dried bacteria stabilization during long-term storage.

Glass transition behavior of carnosine and its impact as a protectant on freeze-dried lactic acid bacteria

Glass transition behavior of carnosine and its impact as a protectant on freeze-dried lactic acid bacteria

Coffee residue as a valorization bio-agent for shelf-life extension of lactic acid bacteria under cryopreservation

The present work describes the feasibility of coffee residue extracts as cryoprotective agents in the storage stability of freeze-dried lactic acid bacteria. Coffee residue extracts were extracted from coffee residue, produced after coffee extraction for coffee powder and instant coffee preparation, using an autoclave. Leuconostoc mesenteroides WiKim32 was selected to evaluate the ability of coffee residue extracts to protect bacteria during freeze-dried storage. The storage stability of freeze-dried Leu. mesenteroides WiKim32 with coffee residue extracts was comparable to those with commercial cryoprotective agents. Coffee residue extracts contributed to storage stability immediately after freeze-drying (61.2%) and subsequent storage (48.7%). Our data indicate that the protective effect of the coffee residue extracts is associated with ions, carbohydrates, and phenolic compounds. Coffee residue extracts are feasible materials, which can reduce the storage and distribution costs compared to commercial agents currently available.

Freeze-Drying of Lactic Acid Bacteria: A Stepwise Approach for Developing a Freeze-Drying Protocol Based on Physical Properties.

Freeze-drying or lyophilization has become a reference process for preserving lactic acid bacteria. The development of stable freeze-dried lactic acid bacteria (LAB) requires maintaining the biological activity of the cells and the macroscopic porous structure while increasing the efficiency of the manufacturing process. Physical properties of protective solutions, such as glass transition and collapse temperatures, are key elements not only for process optimization but also for the stability of freeze-dried LAB. This chapter provides a stepwise approach for developing a protective formulation for thelong-term preservation of LAB and an efficient freeze-drying process. Methods for determining glass transition and collapse temperatures of protective solutions and cell suspensions, as well as water activity and water content of freeze-dried products, are described.

Shelf-life extension of freeze-dried Lactobacillus brevis WiKim0069 using supercooling pretreatment

Shelf-life extension of freeze-dried lactic acid bacteria is important for reducing distribution costs. Herein, effect of cold adaptation prior to freeze drying on the storage stability of freeze-dried Lactobacillus brevis WiKim0069, a kimchi starter, was investigated under supercooling at −5 °C without ice crystal nucleation and formation. Cold adaptation at 10 °C was used as a reference. Cold adaptation caused an increase in surface layer protein and improved cell viability. The surface layer of cold-adapted cells was substantially thicker than that of non-adapted cells, with supercooling inducing the strongest effect. Cold adaptation increased the viability of L. brevis WiKim0069 during and after 56-day storage. After 56 days of storage, the highest viability of 81.2% was observed with supercooling pretreatment. The addition of surface layer protein during cold adaptation further promoted viability, supporting the role of surface layer protein. These results suggest that supercooling pretreatment can be useful for extending the shelf-life of L. brevis WiKim0069.

Probiotics’ effects on perception of health and bowel frequency: a double-blind, randomised, placebo-controlled clinical trial

To evaluate the effects of probiotics on individuals’ perception of health and bowel frequency. The VSL#3 Probiotic (V3P; Visbiome in USA; Vivomixx in EU) contains eight strains of live, freeze-dried lactic acid bacteria. It has some therapeutic potential in ulcerative colitis and pouchitis.

Effect of Cation Influx on the Viability of Freeze-Dried Lactobacillus brevis WiKim0069

Extension of the storage stability of freeze-dried lactic acid bacteria is important for industrialization. In this study, the effect of cation influx from soy powder, which contains high amounts of cations, as a cryoprotective agent on the viability of freeze-dried Lactobacillus brevis WiKim0069 was tested. Compared to that in the absence of the soy powder, bacterial viability was significantly higher in the presence of soy powder. Approximately 4.7% of L. brevis WiKim0069 survived in the absence of the protective agent, whereas 92.8% viability was observed in the presence of soy powder. However, when cations were removed from the soy powder by using ethylenediaminetetraacetic acid (EDTA) and a cationic resin filter, the viability of L. brevis WiKim0069 decreased to 22.9–24.7%. When the soy powder was treated with ethylene glycol tetraacetic acid, the viability was higher than when it was pretreated with EDTA and a cationic resin filter, suggesting that Mg2+ had a role in enhancing the viability of L. brevis WiKim0069. Cold adaptation at 10 °C prior to freeze-drying had a positive effect on the storage stability of freeze-dried L. brevis WiKim0069, with 60.6% viability after 56 days of storage. A decrease in the fluorescence polarization value indicated an increase in membrane fluidity, which regulates the activity of ion channels present in the cell membrane. Cold adaptation caused activation of the cation channels, resulting in increased intracellular influx of cations, i.e., Ca2+ and Mg2+. These results suggest that cold adaptation can be used to improve the storage stability of L. brevis WiKim0069.

The Results of Giardiasis Treatment Using Giardicidal Drugs and their Combination with Probiotics in Children: A Retrospective Cohort Study

Background. Giardiasis in children remains an urgent problem, the significance of which is determined by the endemicity for many countries and regions, outbreaks of sporadic epidemics, polymorphism of clinical symptoms, and insufficient treatment efficacy. Objective. Our aim was to study the results of giardiasis treatment using giardicidal drugs or their combination with probiotics in children. Methods. We analyzed the frequency of Giardia lamblia elimination (the main outcome) as a result of giardiasis treatment (laboratory confirmed) in children aged from 3 months to 18 years who received giardicidal drugs (nifuratel, albendazole) or their combination with probiotics based on Saccharomyces boulardii (probiotic 1) and live freeze-dried lactic acid bacteria Lactobacillus acidophilus (L. gasseri), Bifidobacterium infantis, and Enterococcus faecium (probiotic 2) in outpatient or inpatient settings. Additionally, we registered the duration of the main symptoms of giardiasis (abdominal pain, diarrhea), the prevalence of intestinal dysbiosis and lactase deficiency. The considering period is from January 2015 to September 2017. Results. The results of giardiasis treatment were studied in 4 groups: monotherapy with nifuratel (n = 65) or albendazole (n = 64), a combination of nifuratel + probiotic 1 (n = 67) or albendazole + probiotic 2 (n = 64). The groups were comparable by sex, age, and clinical manifestations of the disease. The elimination of lamblia on the 14–16th day of monotherapy with giardicidal drugs was achieved in 56–60%, when combined with probiotics — in 84% of patients for each combination (df = 3, p < 0.001). Against the background of combination therapy, the disease symptoms (abdominal pain, diarrhea, vomiting) were reversed 1.5 times faster; the number of patients with lactase deficiency and intestinal dysbiosis decreased two and more times, under monotherapy with giardicidal drugs — 1.2 times (df = 3, p < 0.001). Conclusion. Giardiasis treatment in children using combination therapy, including giardicidal drugs and probiotics, is more effective than monotherapy with giardicidal drugs.

유산균의 생존율 향상을 위한 동결건조보호제로서 호박분말의 효과

Food-grade protective agents such as sucrose, skim milk, and pumpkin powder were studied for their ability to improve the viability of freeze-dried lactic acid bacteria, including Lactobacillus plantarum MG989, L. fermentum MG901, Streptococcus thermophilus MG5139, Lactococcus lactis MG534, Enterococcus faecium MG89-2, and Bifidobacterium animalis ssp. lactis MG741. The best results were obtained with 5% sucrose contained 10% skim milk and 2% pumpkin powder; approximately 43.1 to 86.6% cell viability was observed during the freeze-drying process. During accelerated storage of freeze-dried lactic acid bacteria, those survival rate decrease rapidly, reaching 9.8~35.2% in 1 month. As a result, the group (5% sucrose, 10% skim milk, and 2% pumpkin powder) showed the protective effect much higher by 639.5% than the control group (5% sucrose and 10% skim milk). These results suggested that pumpkin powder was good candidate as a potential cryoprotectant agent.

Effect of Protectants on the Survival Rate of Freeze-dried Lactic Acid Bacteria

Effect of Protectants on the Survival Rate of Freeze-dried Lactic Acid Bacteria

Behavior of Escherichia coli O157:H7 and Listeria monocytogenes during fermentation and storage of camel yogurt

In addition to its nutritional and therapeutic properties, camel milk has the ability to suppress the growth of a wide range of foodborne pathogens, but there is a lack of information regarding the behavior of these pathogens in products such as yogurt produced from camel milk. The objective of the current study was to investigate the behavior of Listeria monocytogenes and Escherichia coli O157:H7 during manufacture and storage of camel yogurt. Camel milk inoculated with L. monocytogenes and E. coli O157:H7 was fermented at 43°C for 5h using freeze-dried lactic acid bacteria (LAB) starter cultures (Streptococcus thermophilus and Lactobacillus bulgaricus) and stored at 4 or 10°C for 14 d. Camel milk inoculated with L. monocytogenes and E. coli O157:H7 without starter culture was also prepared. During fermentation, the numbers of L. monocytogenes and E. coli O157:H7 increased 0.3 and 1.6 log cfu/mL, respectively, in the presence of LAB, and by 0.3 and 2.7 log cfu/mL in the absence of LAB. During storage at 4 or 10°C, L. monocytogenes increased 0.8 to 1.2 log cfu/mL by 14d in camel milk without LAB, but in the presence of LAB, the numbers of L. monocytogenes were reduced by 1.2 to 1.7 log cfu/mL by 14 d. Further, E. coli O157:H7 numbers in camel milk were reduced by 3.4 to 3.5 log cfu/mL in the absence of LAB, but E. coli O157:H7 was not detected (6.3 log cfu/mL reduction) by 7d in camel yogurt made with LAB and stored at either temperature. Although camel milk contains high concentrations of natural antimicrobials, L. monocytogenes was able to tolerate these compounds in camel yogurt stored at refrigerator temperatures. Therefore, appropriate care should be taken during production of yogurt from camel milk to minimize the potential for postprocess contamination by this and other foodborne pathogens.

Dynamic modeling of the secondary drying stage of freeze drying reveals distinct desorption kinetics for bound water

ABSTRACTIn freeze drying, the desorption step for reaching a low target moisture content may take a significant fraction of the total process duration. Because the long-term stability of freeze-dried biological products strongly depends on the current moisture content, modeling the desorption process may help safely optimize the secondary drying step. Most published models assume a first-order desorption kinetic, but experimental evidence shows that strongly bound water in the monolayer takes a much longer time to be desorbed than less bound water in multilayer. The proposed model for desorption of freeze-dried lactic acid bacteria preparation accounts for monolayer and multilayer water state in the solid matrix, with very different desorption kinetics. Results showed that the ratio of characteristic desorption times (monolayer/multilayer) was almost 30. Temperature dependence was adequately described by an Arrhenius law in the range of 15 to 40°C. Model parameter identification used simultaneously gravimetric measurements with high time resolution and direct Karl-Fisher titration, from several experiments at different, time-varying temperatures.

유산균 생존율 향상을 위한 식품첨가물 등급의 동결보호제 탐색

김치제조가 미생물첨가에 따른 발효조절로 패러다임이 변화함에 따라 김치에서 분리한 유용 미생물의 분말형 제제 기술을 위한 식품첨가등급의 우수한 보호제를 탐색하고자 하였다. 모든 실험균주에서 보호제를 사용할 경우 무처리구보다 높은 생존율을 나타내었고, 특히 10% 콩가루를 동결보호제로 사용하였을 때 90% 수준의 높은 보호효과를 나타내었다. 콩가루의 농도를 증가시킨 경우 동결건조 후 실험균주의 생존율은 농도 의존적으로 증가하지 않았다. 두 가지 보호제를 1:1의 비율로 조합하여 적용할 경우 생존율 향상에 관한 상승효과는 관찰되지 않았다. Food-grade protective agents, namely, skim milk, yeast extract, soy powder, and trehalose, were studied for their ability to improve the viability of freeze-dried lactic acid bacteria (LAB), including Weissella cibaria SW1-1, Lactobacillus plantarum A-1, Lactobacillus sakei 2-12 24, and Leuconostoc citreum 3526. The best results were obtained with 10% soy powder; approximately 90% cell viability was observed during the freeze-drying process. Increase in the concentration of soy powder did not cause a proportional increase in the survival rate of LAB. Further, no significant difference was observed when two agents were combined in a 1:1 ratio (p<0.05).

Biomass production and small-scale testing of freeze-dried lactic acid bacteria starter strains for cassava fermentations

Based on their predominance in Gari fermentations, as well as suitable technological properties, Lactobacillus plantarum, Lactobacillus fermentum, Weissella paramesenteroides and Leuconostoc mesenteroides strains were investigated for their suitability for development as starter strains for this African traditional fermented cassava product. The strains were grown in optimized growth media in 2 L fermenters, harvested and freeze dried, and then tested in lab-scale cassava mash fermentation trials for their ability to ferment the cassava. The strains performed well and rapidly increased the titratable acidity from 1.1 to 1.3% at 24 h to 1.3–1.6% at 48 h. The benefit of including starter cultures was that it lowered the pH of the product much faster and to lower levels than in the uninoculated control fermentation. The results furthermore indicated that especially the L. plantarum-group strains could be produced as starter strains at low cost. Overall, the results of this study showed that starter strains could be easily and economically produced, and thus represent a feasible possibility for further development for application in the field.

Production of freeze-dried lactic acid bacteria starter culture for cassava fermentation into gari

Sixteen lactic acid bacteria, eight Lactobacillus plantarum, three L. pentosus, 2Weissella paramesenteroides, two L. fermemtum and one Leuconostocmesenteroides ssp. mesenteroides were previously isolated from cassava fermentation and selected on the basis of their biochemical properties with a view to selecting appropriate starter cultures during cassava fermentation for gariproduction. In this study, the potential of these pre-selected strains as suitable freeze-dried cultures was evaluated. Their ability to tolerate the freeze-drying process was assessed by dehydration in a glycerol solution of increasing concentration, followed by staining with two fluorescent markers: rhodamine 123 and propydium iodide. Twelve strains that recovered more than 50% of their population value after visualisation on an epi-fluorescent microscope were produced in a bioreactor and freeze-dried. The technological characteristics identified after the freeze-drying process, were a high cell concentration or high survival rate. The ability of the freeze-dried strains to recover their acidification activity was evaluated through the determination of the pH, titratable acidity (% lactic acid/g Dry Weight) and cell count over 24 h on MRS broth. Ultimately, the strains L.plantarum VE36, G2/25, L. fermentum G2/10 and W. paramesenteroides LC11 were selected to be developed as freeze-dried starter cultures for gari production. Key words: Freeze-drying, gari, lactic acid bacteria, fermentation, starter culture, cassava, developing countries, fermented food.

Survival Rate Analysis of Freeze-Dried Lactic Acid Bacteria Using the Arrhenius and z-Value Models

The survival rate of five freeze-dried bacteria species, Lactobacillus plantarum, Lactobacillus pentosus, Weisella paramesenteroides, Leuconostoc mesenteroides, and Lactobacillus fermentum, was described in terms of reaction rate constants (D or k) and temperature sensitivity of rate constants (z or Ea). The freeze-dried strains were stored under vacuum at 55, 37, and 4°C for 168 h, 17 days, and 2 months, respectively. D-values decreased and k increased with an increase of the storage temperature. Neither the z-value nor the inactivation energy (Ea) of the reaction was significantly different (P > 0.05) for all the strains, suggesting that thermal inactivation of the freeze-dried lactic acid bacteria may occur by the same mechanism. Therefore, it was possible to compare rate constants of survival for the freeze-dried strains studied.

Relevant factors for the preparation of freeze-dried lactic acid bacteria

Abstract The industrial exploitation of lactic acid bacteria (LAB) as starter and/or probiotic cultures depends strongly on the preservation technologies employed, which are required to guarantee long-term delivery of stable cultures in terms of viability and activity. Freeze-dried preparations exhibit advantages relative to preparations made with other techniques in terms of long-term preservation, coupled with convenience in handling, storage, marketing and application. Degrees of survival of LAB cultures as high as possible, during drying and subsequent storage, are thus of nuclear importance, both technologically and economically. This review covers several factors, deemed relevant for preservation of freeze-dried LAB. The state of the art of the knowledge focussed on the improvement of LAB survival during freeze-drying and subsequent storage is presented, including specific discussion of the effects of (i) intrinsic factors, (ii) growth factors, (iii) sub-lethal treatments, (iv) drying media and (v) storage and rehydration.