Microbial Biotechnology (MB) | Cell Culture and Biomedical Engineering
Microbiol. Biotechnol. Lett. 2021; 49(2): 157-166
https://doi.org/10.48022/mbl.2012.12007
Karina Arellano1†, Haryung Park2†, Bobae Kim2, Subin Yeo2, Hyunjoo Jo3, Jin-Hak Kim4, Yosep Ji3, and Wilhelm H. Holzapfel1,2*
1Department of Advanced Convergence, 2HEM Inc., Business Incubator Center 103, Handong Global University, Pohang 37554, Republic of Korea 3HEM Inc., 404, Ace Gwanggyo Tower 3, Suwon 16229, Republic of Korea 4COSMAX NS Inc., Seongnam 13486, Republic of Korea
Correspondence to :
Wilhelm Holzapfel, wilhelm@woodapple.net
The probiotic market is constantly continuing to grow, concomitantly with a widening in the range and diversity of probiotic products. Probiotics are defined as live microorganisms that provide a benefit to the host when consumed at a proper dose; the viability of a probiotic is therefore of crucial importance for its efficacy. Many products undergo lyophilization for maintaining their shelf-life. Unfortunately, this procedure may damage the integrity of the cells due to stress conditions during both the freezing and (vacuum-) drying process, thereby impacting their functionality. We propose a lysine-based mixture for rehydration of freeze-dried probiotics for improving their viability during in vitro simulated gastric and duodenum stress conditions. Measurement of the zeta potential served as an indicator of cell integrity and efficacy of this mixture, while functionality was estimated by adhesion to a human enterocyte-like Caco-2 cell-line. The freeze-dried bacteria exhibited a significantly different zeta potential compared to fresh cultures; however, this condition could be restored by rehydration with the lysine mixture. Recovery of the surface charge was found to influence adhesion ability to the Caco-2 cell-line. The optimum lysine concentration of the formulation, designated “Zeta-bio”, was found to be 0.03 M for improving the viability of Lactiplantibacillus plantarum Lp-115 by up to 13.86% and a 7-strain mixture (400B) to 41.99% compared to the control rehydrated with distilled water. In addition, the lysine Zeta-bio formulation notably increased the adherence ability of lyophilized Lp-115 to the Caco-2 cell-line after subjected to the in vitro stress conditions of the simulated gastrointestinal tract passage.
Keywords: Zeta potential, probiotic, viability, cell adhesion, L-lysine
Probiotics are defined to have a beneficial impact on host health when administered in adequate amounts. Scientific evidence is steadily accumulating on the beneficial impact of probiotics on human health in various ways including the alleviation of immune disorders, inflammatory bowel disease, type 2 diabetes and atherosclerosis [1−4]. Although recommendations tend to favor the consumption of high dose probiotics, neither the specific dosage nor the minimal viable numbers required for a putative probiotic strain are well-defined [5]. Strains with potential probiotic properties can be “naturally” obtained via fermented food such as fermented dairy products, yet the distribution of freeze-dried probiotic powders packaged in sachets or capsules are rapidly expanding in the market [6]. Marketed probiotics should be transportable, shelf-stable concentrates that guarantee the effects of intrinsic functional properties [7]. Commercialization of non-dairy probiotic products requires exact optimization of the final processing steps such as the harvesting, freezing and drying process [8]. The freeze-drying (a.k.a. lyophilization) process is known to be a stressful condition for live bacteria, yet this technology is still considered an appropriate approach to guarantee an extended shelf life for most probiotic products. The freeze-drying process is indeed a “challenge” to the viability of probiotic strains. Therefore, in order to maintain an effective dose, the number of bacteria in most products are generally three- to ten-fold higher compared to the numbers stated on the product label [5].
Key requirements for an effective probiotic include its survival under gastrointestinal stress conditions and its adhesion to the intestinal wall. Colon cell-lines may be used as an
The electrostatic charge of the cell surface is considered to be a reflection of its functional groups. When in contact with a liquid the surface charge of a bacterial cell can be measured in millivolt units as zeta or electrokinetic potential; values for living cells are typically more negative than those for dead cells [15]. Both the cell surface composition and the properties of the surrounding medium (e.g., conductivity/ionic strength and pH) will determine the cell’s zeta potential [16]. Changes in the zeta potential (caused, e.g., by cationic agents) reflect membrane damage and alterations in permeability and may thus serve to predict cell viability [17]. Composition and chemical characteristics of the cell surface play a key role in its response to the surrounding medium (e.g., conductivity/ionic strength and pH) and thereby in determining the electrostatic charge of the cell surface and its zeta potential [16]. Zeta potential can therefore be used as a measure of the electrical surface charge of a bacterial particle in a suspension [18]. It is determined by the nature of the groups exposed at the surface, and under normal physiological conditions bacteria are usually negatively charged due to the large amount of phosphate and carboxyl groups present on the cell surface [19]. Together with interfacial viscoelasticity and tension the zeta potential can serve to characterize the surface properties of intestinal bacteria and to predict their adhesion potential in the GI tract [20].
The zeta potential around bacteria can thus serve as an indicator of their viability, integrity and efficacy especially in terms of overall and physiological potential [21]. We therefore propose a new approach for sachet packaged probiotics containing the probiotic together with a special lysine-based formulation for supporting the re-activation of a probiotic strain in water. We have named this mixture “Zeta-bio” since the zeta potential was used as an initial screening factor for 33 different chemicals using three representative strains [(
All strains and freeze-dried probiotics including COSMAX NBT (400B) 7-strain HRB and its materials (Table 1) were provided by COSMAX NBT Inc. (Korea). Each probiotics sachet contained 3 × 1010 CFU of the freeze-dried microorganism mix with 3.5 g of fructooligosaccharides (FOS) and dextrin (Table 1).
Table 1 . Concentrations of ingredients combined for the potential Zeta-bio formulation.
Ingredients (Brand) | LP/Mix-1 | LP/Mix-2 | LP/Mix-3 | LP/Mix-4 | LP/Mix-5 |
---|---|---|---|---|---|
L-lysine hydrochloride (Samin) | 0.182 g (0.01 M) | 0.364 g (0.02 M) | 0.546 g (0.03 M) | 0.728 g (0.04 M) | 0.910 g (0.05 M) |
FOS (Hi-tech) | 3.5 g | 3.5 g | 3.5 g | 3.5 g | 3.5 g |
Microorganism | 0.15 g (2 × 1011 CFU/g) | 0.15 g (2 × 1011 CFU/g) | 0.15 g (2 × 1011 CFU/g) | 0.15 g (2 × 1011 CFU/g) | 0.15 g (2 × 1011 CFU/g) |
Dextrin (Daesang) | 6.204 g | 6.130 g | 5.912 g | 5.766 g | 5.62 g |
Total | 10 g | 10 g | 10 g | 10 g | 10 g |
The lyophilized probiotic powders of
Table 2 . List of ingredients tested for their zeta potential activity.
Ingredient | Chemical formula | Molecular weight (g/mol) | Purity (%) | Brand | Lot no. |
---|---|---|---|---|---|
Carbohydrates | |||||
Arabinose | C5H10O5 | 150.13 | 100 | USB | 70047 |
Xylose | C5H10O5 | 150.1 | 99 | Sigma | 053K00131 |
Rhamnose | C6H12O5. H20 | 182.2 | 99 | Sigma | 058K0695 |
Fructose | C6H12O6 | 180.16 | 100 | Sigma | 125K01611 |
Mannitol | C6H14O6 | 182.17 | 100 | Sigma | 034K0061 |
Sucrose | C12H22O11 | 342.3 | 100 | Daejung | S0784RE1 |
Sorbitol | C6H14O6 | 182.17 | 97 | Daejung | S0503QC1 |
Glucose | C6H12O6 | 180.16 | 99.5 | Sigma | SLBS2877V |
Maltose | C12H22O11.H2O | 360.31 | 100 | USB | 109469 |
Trehalose | C12H22O11.2H20 | 378.33 | 99 | Sigma | SLBV3123 |
Fructooligosaccharide | C6H12O6 | - | 95 | Qhtbio | 10002428 |
Amino acids | |||||
L-Arginine | C6H14N4O2 | 174.2 | 98 | Sigma | 017K0664 |
L-Tryptophan | C11H12N2O2 | 204.23 | 99 | Kanto chemical co. | 403N2187 |
L-Phenylalanine | C9H11NO2 | 165.19 | 99 | Junsei | 2012L1433 |
L-Ornithine | C5H12N2O2.ClH | 168.62 | 99 | Sigma | 109K1468 |
L-Glutamic acid | C5H8NNaO4.xH2O | 169.11 | 99 | Sigma | SLBF7449V |
L-Proline | C5H9N02 | 115.1 | 100 | Sigma | 72H0774 |
L-Lysine hydrochloride | C6H14N2O2.HCl | 182.65 | 98 | Sigma | 108K1321 |
L-Serine | C3H7NO3 | 105.09 | 99 | Georgiachem | S454621R |
L-Threonine | C4H9NO3 | 119 | 99 | Georgiachem | T262958H |
L-Aspartic acid | C4H7NO4 | 133.1 | 99 | Georgiachem | A977123A |
L-Tyrosine | C9H11NO3 | 181.19 | 99 | Samchun chemicals | 101917 |
L-Histidine | C6H9N3O2 | 209.64 | 99 | Daejung | H2821RL1 |
Salts | |||||
Sodium phosphate | Na2HPO4 | 141.96 | 99 | Sigma | 075K2520 |
Sodium L-tartrate dihydrate | C4H4Na2O6.2H2O | 230.08 | 99 | Sigma | 07425BC |
Sodium bicarbonate | NaHCO3 | 84 | 99.8 | Yakuri | 312132812 |
Organic acids | |||||
Malic acid | C4H6O5 | 134 | 98 | Sigma | SLBS7651 |
Pyruvic acid | C3H4O3 | 80.06 | 100 | Daejung | P3470SG1 |
Osmolytes | |||||
Betaine | C5H11NO2 | 117 | 98 | Daejung | B1549PE1 |
Taurine | C2H7NO3S | 125.15 | 99 | Sigma | 12515DU |
Vitamins | |||||
Riboflavin | C17H20N4O6 | 376.37 | 100 | Sigma | 069k1585 |
Thiamine hydrochloride | C12H17ClN4OS.HCl | 337.27 | 98 | Daejung | T0027QL1 |
L-Ascorbic acid | C6H8O6 | 176.12 | 99 | TCI | KNBGE |
The potential of Zeta-bio formulations (Table 1) for the activation, adhesion and protection of freeze-dried
Measurement of the zeta potential was used to screen the effect of 33 chemical ingredients on three representative bacterial strains of the 7-strain mixture (400B):
Freshly cultivated and freeze-dried dried bacterial suspensions of each strain were used as positive and negative controls to compare the effect of lysine and proline on the cell adhesion of the freeze-dried bacteria. Freeze-dried
Based on the analysis of viability and cell adhesion, the two key parameters that are known to be influenced by the zeta potential, the optimal concentration of lysine was selected. The concentration of the lysine-based Zetabio formula was reduced to 0.01−0.05 M, considering the taste effect for the final product formulation (Table 1). Further analysis was conducted using the actual commercial products: 7-strain mixture (400B) and the single cell probiotic,
SSDP was performed to check the effect of the different concentrations of lysine in the Zeta-bio formulation on the viability of the freeze-dried bacteria after simulated passage of the stomach and duodenum. The viability of the freeze-dried form of
Table 3 . Effects of lysine concentration in the Zeta-bio formulation on survival during simulated stomach-duodenum passage (SSDP).
Sample name | Strain | Lysine concentration | Initial | Stomach | Duodenum | ||
---|---|---|---|---|---|---|---|
log CFU/ml | log CFU/ml | Survival (%) | log CFU/ml | Survival (%) | |||
Lp-115 fresh | none | 8.21±0.10 | 8.05±0.26 | 64.45 | 7.61±0.15 | 25.76 *** | |
Lp-115 FD | none | 8.04±0.16 | 4.15±0.16 | 0.01 | 4.02±0.66 | 0.02 | |
LP-1 | 0.01M | 7.05±0.21 | 5.63±0.05 | 5.07 | 4.29±0.28 | 0.17 *** | |
LP-2 | 0.02M | 8.46±0.04 | 6.97±0.01 | 4.25 | 6.26±0.01 | 0.63 *** | |
LP-3 | 0.03M | 8.59±0.03 | 8.28±0.08 | 48.64 | 7.73±0.01 | 13.86 *** | |
LP-4 | 0.04M | 8.62±0.02 | 8.30±0.00 | 48.01 | 7.69±0.00 | 11.93 *** | |
LP-5 | 0.05M | 8.63±0.09 | 8.26±0.01 | 43.20 | 7.72±0.03 | 12.17 *** | |
Mix FD | 7 strain mixture (400B) | none | 8.75±0.02 | 7.52±0.05 | 6.01 | 4.96±0.07 | 0.02 |
Mix-1 | 0.01M | 8.20±0.08 | 6.43±0.00 | 1.74 | 5.88±0.06 | 0.48 *** | |
Mix-2 | 0.02M | 8.42±0.01 | 7.79±0.01 | 23.02 | 7.69±0.04 | 18.70 ** | |
Mix-3 | 0.03M | 8.32±0.01 | 8.02±0.01 | 50.09 | 7.94±0.05 | 41.99 ** | |
Mix-4 | 0.04M | 8.33±0.03 | 7.95±0.03 | 41.93 | 7.90±0.01 | 37.65 * | |
Mix-5 | 0.05M | 8.37±0.01 | 8.10±0.01 | 53.33 | 7.82±0.14 | 28.72 * |
The data are shown as mean ± standard deviation. Statistical analysis for the Lp-115 was performed by student’s
The different lysine concentrations strongly influenced the adherence ability of the freeze-dried bacteria to the intestinal Caco-2 cell-line. Compared to the freshly cultivated culture the freeze-dried cells of
Preserving the integrity of the cell envelope, and particularly of the plasma membrane, is important in maintaining cell viability and homeostasis. Adhesive potential may be considered as a criterion for reflecting functionality of an effective potential probiotic. Successful survival after gastric stress and proper attachment to the intestinal cells will promote immunomodulatory and metabolic functions, and thereby serve to strengthen the gut barrier and competitively inhibit the adhesion of pathogens [24]. Key adhesive factors on the cell surface of Gram-positive bacteria include lipoteichoic acid, surface layer proteins and mucous binding proteins [25]. Although the process of lyophilization helps to maintain bacterial shelf-life by the reduction of water activity, it also may disrupt the cell membrane thereby leading to the loss of its original functionality [26]. Recovering the integrity of the cell membrane may help reactivate its functionality through increase in viability and cell adhesive properties.
The depolarization of the zeta potential has been suggested as a positive indicator of bacterial cell membrane damage [17, 21]. Therefore, 33 ingredients were tested for their effect on the zeta potential of all three different strains. Deepika
Further research was performed to find the optimal concentration of L-lysine for the Zeta-bio formulation which is mixed with the prebiotic fructooligosaccharides (FOS). Besides enhancing the taste of the product, FOS is a well-known and commonly used prebiotic added for its prebiotic advantage and for improving the viability of probiotics due to its resistance to gastric stress [30−33]. Moreover, FOS is also known to provide beneficial health effects through the stimulation of colon bacteria producing short chain fatty acids, and for reducing weight-gain and preventing intestinal diseases [34]. FOS is also known to modulate the microbiome to a healthy state by increasing the ratio of the commensal bacteria in the human gut [35]. Furthermore, the addition of L-lysine to FOS is known to help enhance the resistance of a strain under unbalanced osmotic conditions [36].
When the Zeta-bio formulation with different lysine concentrations (0.01 M−0.05 M) was applied to the commercial probiotic powders
In conclusion, the application of the 0.03 M lysinebased Zeta-bio formulation for freeze-dried probiotic products, as exemplified by
The authors have no financial conflicts of interest to declare.
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