Cell immobilization techniques for the preservation of probiotics

Incorporation of probiotic cultures in products in order to replenish or supplement the normal gastrointestinal microflora is a well known and accepted practice. However survival of these cultures is a problem due to a number of reasons including effects of storage conditions. Various researchers from different countries around the world have reported probiotic product instability. Microencapsulation has been used in an attempt to solve this problem. However, most methods involve the use of organic solvents which is not ideal because their toxicity may cause destruction of the microbial cells. A novel encapsulation method for probiotics, which excludes the use of organic solvents, was developed by the Council for Scientific and Industrial Research (CSIR) (US Patent Application no. 20050112205). This thesis investigated the efficiency/potential of this new method for increasing stability of sensitive probiotic cultures, specifically bifidobacteria. Early studies using both culture dependent and culture independent techniques showed reduced numbers of viable cultures in probiotic products, mainly yoghurts, from all around the world. These results were confirmed in this study for similar products sold in South Africa. Most of the product labels did not specify viable numbers of probiotics nor the identity (genus and species names) of the microorganisms incorporated. Successful encapsulation of bifidobacteria was achieved using the CSIR patented method. Complete encapsulation was indicated by absence of cells on surfaces of the encapsulated particles and production of a product with an acceptable particle size distribution was obtained. It was also demonstrated that the encapsulation process produced no visible morphological changes to the bacterial cells nor did it have a negative effect on cell viability over time. The potential of interpolymer complex formation in scCO2 for the encapsulation of sensitive probiotic cultures was demonstrated for the first time. Once ingested, probiotic cultures are exposed to unfavourable acidic conditions in the upper gastrointestinal tract. It is desired that these cultures be protected from this in order to increase the viability of the probiotics for efficient colonization. Interpolymer complex encapsulated B. longum Bb-46 cells were therefore exposed to simulated gastric fluid (SGF) and subsequently to simulated intestinal fluid (SIF). It was found that the interpolymer complex protected bifidobacteria from gastric acidity, displaying pH-responsive release properties, with little to no release in SGF and substantial release in SIF. Thus the interpolymer complex demonstrated desirable characteristics retaining the encapsulated bacteria inside when conditions were unfavourable and only releasing them under favourable conditions. Survival was improved by the incorporation of glyceryl monostearate (GMS) in the matrix and by use of gelatine capsules. Protection efficiency of the interpolymer matrix was better when higher loading of GMS was used. Use of polycaprolactone (PCL) as an alternative to poly (vinylpyrrolidone) (PVP) and incorporation of ethylene oxide-propylene oxide triblock copolymer (PEO-PPO-PEO) affected the interpolymer complex negatively, rendering it swellable in the low pH environment exposing the bifidobacteria to gastric acidity. The use of beeswax seemed to have a more protective effect though results were inconclusive. Probiotic cultures must also remain viable in products during storage. Encapsulated bacteria were either harvested from the reactor after 2 h of equilibration followed by depressurization, and then ground to a fine powder or after 2 h of equilibration the liquefied product was sprayed through a capillary tube with a heated nozzle at the end, into the product chamber. Encapsulated bacteria were stored in either sterile plastic bags or glass bottles under different conditions and then viable counts were determined over time. Survival of bacteria was generally better when the products were stored in glass bottles than in plastic bags. Bacteria encapsulated in an interpolymer complex formed between PVP and vinyl acetate-crotonic acid copolymer (VA-CA), (PVP:VA-CA) survived better than non-encapsulated bacteria under all storage conditions when the product was recovered from the reaction chamber. When the product was recovered from the product chamber, numbers of viable non-encapsulated bacteria were higher than the encapsulated bacteria for all interpolymer complex formulations. This was probably due to some exposure to high shear during spraying into the product chamber. The interpolymer complex between PCL and VA-CA i.e. PCL:VA-CA seemed weaker than the PVP:VA-CA nterpolymer complex as viable counts of bacteria released from it were lower than those from the latter complex. Addition of PEO-PPO-PEO to both the PVP:VA-CA and PCL:VA-CA complexes decreased the protection efficiency. However, results indicated that sufficient release of encapsulated bacteria from the interpolymer complexes was obtained when the encapsulated material was incubated in SIF rather than in Ringer’s solution. When SIF was used for release of encapsulated bacteria, the shelf life of B. longum Bb-46 was doubled. Encapsulation in an interpolymer complex therefore provided protection for encapsulated cells and thus has potential for improving shelf life of probiotic cultures in products. Further studies will investigate the effects of encapsulating probiotics together with prebiotics in the interpolymer complex as well as effects of encapsulating combinations of different probiotic strains together, both on survival in simulated gastrointestinal tract and during storage. The unique particles produced using the patented encapsulation technique increased the stability of probiotic cultures. This technique may find significant application in industries manufacturing probiotic products, especially food and pharmaceuticals, thereby improving the well being of consumers.Thesis (PhD(Microbiology))--University of Pretoria, 2008.Microbiology and Plant PathologyPhDunrestricte

Fourier transform infra-red spectroscopy and flow cytometric assessment of the antibacterial mechanism of action of aqueous extract of garlic (Allium sativum) against selected probiotic Bifidobacterium strains

BACKGROUND: It is generally reported that garlic (Allium sativum) harms pathogenic but not beneficial bacteria. Although numerous studies supporting the alleged garlic effects on pathogens are available, there are limited studies to prove this claim for beneficial bacteria. We have recently shown that garlic exhibits antibacterial activity against probiotic bifidobacteria. The aim of the current study was to elucidate the mechanism of action of garlic clove extract (GCE) on Bifidobacterium bifidum LMG 11041, B. longum LMG 13197 and B. lactis Bb12 using Fourier transform infrared (FT-IR) spectroscopy and flow cytometry. METHODS: Cultures (1 × 108 CFU ml-1) were individually incubated for 6 h at 37°C in garlic clove extract containing allicin at a corresponding predetermined minimum bactericidal concentration for each strain. For FTIR, an aliquot of each culture was deposited on CaF2 slide and vacuum dried. The slides were immediately viewed using a Bruker Vertex 70 V FT-IR spectrometer equipped with a Hyperion microscope and data analyzed using OPUS software (version 6, Bruker). Spectra were smoothed with a Savitsky-Goly function algorithim, base-line corrected and normalized. Samples for flow cytometry were stained using the Live/Dead BacLight bacterial viability kit L7012. Data compensation and analysis was performed using a BD FACSAria and FlowJo (version 7.6.1). RESULTS: Fourier transform infrared spectroscopy showed changes in spectral features of lipids and fatty acids in cell membranes, proteins, polysaccharides and nucleic acids. Spectral data as per principle component analysis (PCA) revealed segregation of control and GCE-treated cells for all the tested bifidobacteria. Flow cytometry not only showed increase in numbers of membrane damaged and possibly lysed cells after GCE treatment, but also displayed diffuse light scatter patterns for GCE treated cells, which is evidence for changes to the size, granularity and molecular content of the cells. CONCLUSION: Garlic has multiple target sites in bifidobacteria, penetrating the cell membrane and entering the cytoplasm, where it causes changes to carbohydrates, fatty acids, proteins and nucleic acids. These changes, for example, modification of membrane properties, may prevent exposed bifidobacteria from colonizing the intestinal mucosa. Loss of colonization potential would render them less efficient as probiotics.The National Research Foundation and University of Pretoriahttp://www.biomedcentral.com/bmccomplementalternmedam201

Antibacterial effect of hydrosoluble extracts of garlic (Allium sativum) against Bifidobacterium spp. and Lactobacillus acidophilus

The antimicrobial effects of garlic (Allium sativum) against pathogenic microorganisms have been well documented. It is generally stated that garlic exhibits differential inhibition between pathogenic and beneficial bacteria. Though there is substantial evidence to support the claim for pathogens, there is limited literature on its effects on beneficial bacteria, specifically probiotic bifidobacteria. This study aimed to investigate the antimicrobial effects of different garlic preparations on five strains of bifidobacteria. The disk diffusion assay revealed antibacterial activity of different garlic preparations characterised by zones of inhibition ranging from 13.0 ± 1.7 to 36.7 ± 1.2 mm. Minimum inhibitory concentration (MIC) values for garlic clove extract ranged from 75.9 to 303.5 mg/ml (estimated 24.84 to 99.37 μg/ml allicin). Bifidobacterium lactis Bi-07 300B was on average the most resistant to garlic, followed by B. lactis Bb12, B. longum LMG 13197, B. longum Bb356 and B. bifidum 11041, being most sensitive. This study reveals for the first time, susceptibility of bifidobacteria to antibacterial activity of garlic. Caution is therefore advised when using probiotic bifidobacteria and garlic simultaneously.The National Research Foundation (NRF) and the University of Pretoria.http://www.academicjournals.org/AJMRam201

Anti-biofilm activity of cell free supernatants of selected lactic acid bacteria against listeria monocytogenes isolated from avocado and cucumber fruits, and from an avocado processing plant

DATA AVAILABILITY STATEMENT : Data are contained within the article.Listeria monocytogenes forms biofilms on food contact surfaces, a niche from where it dislodges to contaminate food products including fresh produce. Probiotics and their derivatives are considered promising alternative strategies to curb the presence of L. monocytogenes in varied food applications. Nonetheless, studies on their anti-biofilm effects against L. monocytogenes from avocados and cucumbers are sparse. This study screened the biofilm formation capabilities of L. monocytogenes strains Avo and Cuc isolated from the avocado and cucumber fruits respectively, and strain 243 isolated from an avocado processing plant; and evaluated the anti-biofilm effects of cell free supernatants (CFS) of Lactobacillus acidophilus La14 150B, Lactiplantibacillus plantarum B411 and Lacticaseibacillus rhamnosus ATCC 53103 against their biofilms formed on polyvinyl chloride (PVC) and stainless steel. All the L. monocytogenes strains formed biofilms (classified either as moderate or strong biofilm formers) on these materials. The presence of CFS reduced the biofilm formation capabilities of these strains and disrupted the integrity of their pre-formed biofilms. Quantitative reverse transcriptase polymerase chain reaction revealed significant reduction of positive regulatory factor A (prfA) gene expression by L. monocytogenes biofilm cells in the presence of CFS (p < 0.05). Thus, these CFS have potential as food grade sanitizers for control of L. monocytogenes biofilms in the avocado and cucumber processing facilities.The University of Pretoria and the National Research Foundation of South Africa.https://www.mdpi.com/journal/foodsam2023BiochemistryGeneticsMicrobiology and Plant Patholog

Cocktails of probiotics pre-adapted to multiple stress factors are more robust under simulated gastrointestinal conditions than their parental counterparts and exhibit enhanced antagonistic capabilities against Escherichia coli and Staphylococcus aureus

BACKGROUND : The success of the probiotics in delivery of health benefits depends on their ability to withstand the technological and gastrointestinal conditions; hence development of robust cultures is critical to the probiotic industry. Combinations of probiotic cultures have proven to be more effective than the use of single cultures for treatment and prevention of heterogeneous diseases. We investigated the effect of pre- adaptation of probiotics to multiple stresses on their stability under simulated gastrointestinal conditions and the effect of their singular as well as their synergistic antagonistic effect against selected enteric pathogens. METHODS : Probiotic cultures were inoculated into MRS broth adjusted to pH 2 and incubated for 2 h at 37°C. Survivors of pH 2 were subcultured into 2% bile acid for 1 h at 37°C. Cells that showed growth after exposure to 2% bile acid for 1 h were finally inoculated in fresh MRS broth and incubated at 55°C for 2 h. The cells surviving were then used as stress adapted cultures. The adapted cultures were exposed to simulated gastrointestinal conditions and their non- adapted counterparts were used to compare the effects of stress adaptation. The combination cultures were tested for their antipathogenic effects on Escherichia coli and Staphylococcus aureus. RESULTS : Acid and bile tolerances of most of the stress-adapted cells were higher than of the non-adapted cells. Viable counts of all the stress-adapted lactobacilli and Bifidobacterium longum LMG 13197 were higher after sequential exposure to simulated gastric and intestinal fluids. However, for B. longum Bb46 and B. bifidum LMG 13197, viability of non-adapted cells was higher than for adapted cells after exposure to these fluids. A cocktail containing L. plantarum + B. longum Bb46 + B. longum LMG 13197 best inhibited S. aureus while E. coli was best inhibited by a combination containing L. acidophilus La14 150B + B. longum Bb46 + B. bifidum LMG 11041. A cocktail containing the six non- adapted cultures was the least effective in inhibiting the pathogens. CONCLUSION : Multi-stress pre-adaptation enhances viability of probiotics under simulated gastrointestinal conditions; and formulations containing a mixture of multi stress-adapted cells exhibits enhanced synergistic effects against foodborne pathogens.We would like to acknowledge National Research Foundation (NRF) and the University of Pretoria for funding this work.http://www.gutpathogens.comam201

In vitro antibacterial mechanism of action of crude garlic (Allium sativum) clove extract on selected probiotic Bifidobacterium species as revealed by SEM, TEM, and SDS-PAGE analysis

There has been much research on the effects of garlic (Allium sativum) on numerous pathogens, but very few, if any, studies on its effect on beneficial, probiotic bifidobacteria. We have recently shown that garlic exhibits antibacterial activity against bifidobacteria. The mechanism by which garlic kills bifidobacteria is yet to be elucidated. This study sought to determine the mechanism of action of garlic clove extract on selected Bifidobacterium species using scanning and transmission electron microscopy and SDS-PAGE analysis. SEM micrographs revealed unusual morphological changes such as cell elongation, cocci-shaped cells with cross-walls, and distorted cells with bulbous ends. With TEM, observed changes included among others, condensation of cytoplasmic material, disintegration of membranes, and loss of structural integrity. SDS-PAGE analysis did not reveal any differences in whole-cell protein profiles of untreated and garlic clove extract-treated cells. The current study is the first to reveal the mechanism of action of garlic clove extract on probiotic Bifidobacterium species. The results indicate that garlic affects these beneficial bacteria in a manner similar to that exhibited in pathogens. These results therefore further highlight that caution should be taken especially when using raw garlic and probiotic bifidobacteria simultaneously as viability of these bacteria could be reduced by allicin released upon crushing of garlic cloves, thereby limiting the health benefits that the consumer anticipate to gain from probiotics.National Research Foundation (NRF) and the University of Pretoria.http://link.springer.com/journal/12602hb201

Biological degradation of oil sludge : a review of the current state of development

Oil sludge is a thick viscous mixture of sediments, water, oil and hydrocarbons, encountered during crude oil refining, cleaning of oil storage vessels and waste treatment. Polycyclic aromatic hydrocarbons (PAHs), which are components of crude oil sludge, constitute serious environmental concerns, as many of them are cytotoxic, mutagenic and potentially carcinogenic. Improper management and disposal of oil sludge causes environmental pollution. The adverse effects of oil sludge on soil ecology and fertility have been of growing interest among environmental scientist and an important consideration in the development of efficient technologies for remediation of contaminated land, with a view to making such land available for further use. Oil sludge can be treated by several methods such as physical, chemical and biological processes. The biological processes are mostly cost effective and environmentally friendly, as they are easy to design and implement, as such they are more acceptable to the public. Compost, the product of biological breakdown of organic matter is a rich source of hydrocarbon-degrading microorganisms such as bacteria and fungi. These microorganisms can degrade the oil sludge to less toxic compounds such as carbon dioxide, water and salts. Compost bioremediation, the application of composting in remediation of contaminated environment, is beginning to gain popularity among remediation scientists. The success or failure of compost bioremediation depends on a number of factors such as nutrients, pH, moisture, aeration and temperature within the compost pile. The bioavailability and biodegradability of the substrate to the degrading microorganisms also contributes to the success of the bioremediation process. This is a review on the biological remediation technologies employed in the treatment oil sludge. It further assesses the feasibility of using compost technology for the treatment of oil sludge, as a better, faster and more cost effective option.The South African National Research Foundationhttp://www.academicjournals.org/AJBam201

Supercritical CO2 interpolymer complex encapsulation improves heat stability of probiotic bifidobacteria

The probiotic industry faces the challenge of retention of probiotic culture viability as numbers of these cells within their products inevitably decrease over time. In order to retain probiotic viability levels above the therapeutic minimum over the duration of the product’s shelf life, various methods have been employed, among which encapsulation has received much interest. In line with exploitation of encapsulation for protection of probiotics against adverse conditions, we have previously encapsulated bifidobacteria in poly-(vinylpyrrolidone)-poly-(vinylacetate-co-crotonic acid) (PVP:PVAc-CA) interpolymer complex microparticles under supercritical conditions. The microparticles produced had suitable characteristics for food applications and also protected the bacteria in simulated gastrointestinal fluids. The current study reports on accelerated shelf life studies of PVP:PVAc-CA encapsulated Bifidobacterium lactis Bb12 and Bifidobacterium longum Bb46. Samples were stored as free powders in glass vials at 30 °C for 12 weeks and then analysed for viable counts and water activity levels weekly or fortnightly. Water activities of the samples were within the range of 0.25–0.43, with an average a w = 0.34, throughout the storage period. PVP:PVAc-CA interpolymer complex encapsulation retained viable levels above the recommended minimum for 10 and 12 weeks, for B. longum Bb46 and B. lactis Bb12, respectively, thereby extending their shelf lives under high storage temperature by between 4 and 7 weeks. These results reveal the possibility for manufacture of encapsulated probiotic powders with increased stability at ambient temperatures. This would potentially allow the supply of a stable probiotic formulation to impoverished communities without proper storage facilities recommended for most of the currently available commercial probiotic products.University of Pretoria, National Research Foundation (NRF), South Africa and The Council for Scientific and Industrial Research (CSIR), Pretoria.http://www.springer.com/chemistry/biotech/journal/11274hb201

Comparison of different methods for release of Bifidobacterium longum Bb46 from the poly(vinylpyrrolidone)-poly(vinylacetate-co-crotonic acid) interpolymer complex matrix, and the effect of grinding on the microparticles

Bifidobacteria have been efficiently encapsulated in poly(vinylpyrrolidone)-poly(vinylacetate-co-crotonic acid) (PVP:PVAc-CA) interpolymer complex formed in scCO2. Research indicated that this method improves the stability of encapsulated bacteria in simulated gastrointestinal fluids in vitro. However, further analysis indicated release of lower numbers of encapsulated bacteria from the encapsulating matrix. The aims of this study were to determine a method that would release high numbers of bacteria from the PVP:PVAc-CA interpolymer complex matrix microparticles, and furthermore, to determine the effects of milling on the morphological properties of the microparticles. Three release methods, namely sonication, homogenization in a stomacher and incubation in simulated intestinal fluid (SIF) were compared. Released viable bacteria were assayed using plate counts. Viable bacteria released using a stomacher were three orders of magnitude higher than those released by incubation and an order of magnitude higher than those released using sonication. SEM indicated no negative effects such as exposure of encapsulated bacteria on the matrix due to milling of product. Homogenization in a stomacher is the most efficient method for releasing bacteria from the PVP:PVAc-CA interpolymer complex matrix. Particle size of the PVP:PVAc-CA microparticles encapsulating bacteria can be reduced further by grinding, without exposing the enclosed bacteria.The authors would like to thank the National Research Foundation of South Africa for funding of the project.http://www.springer.com/chemistry/biotech/journal/1127

Isolation, identification and screening of potential probiotic bacteria in milk from South African Saanen goats

This study aimed to evaluate lactic acid bacteria isolates from Saanen goats’ milk for probiotic attributes, thereby determining their potential as direct-fed microbials for goats. Isolates were identified using API 50CH system, 16S rDNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry. All 17 isolates obtained were identified as Lactobacillus plantarum except one identified as Pediococcus acidilactici. Four isolates identified as L. plantarum (Accession numbers KJ026587.1, KM207826.1, KC83663.1 and KJ958428.1) by at least two of the techniques used and isolate 17 differently identified by all the methods used were selected as representatives and then screened for probiotic properties. These isolates displayed phenotypic probiotic attributes including tolerance to acid and bile salts, ability to adhere to intestines and possession of antagonistic activities against Proteus vulgaris, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa and Escherichia coli. The lactic acid bacteria isolated from Saanen goats’ milk showed potential to be used as sustainable probiotics in goats’ industry. Successful use of probiotics in animals depends upon availability of appropriate isolates originating from the specific host animal. This study is a positive contribution towards identification of isolates with potential for formulation as direct-fed microbials for South African Saanen goats.The Agricultural Research Council, Irene, South Africahttp://link.springer.com/journal/126022018-09-01hb2017Microbiology and Plant Patholog