Lactobacillus is the largest genus of lactic acid bacteria (LAB). It has a diverse distribution and accounts for the majority of isolated lactic acid bacteria, originating mainly from the intestinal tract of humans, animals, plants, food and even in muddy environments, aquaculture water. but also showed great phenotypic, biochemical and physiological variation in strains from different isolates. Lactobacillus sp. considered to be a common strain of bacteria commonly used in the fermentation of foods such as cheese, yogurt, sourdough bread, silage, olives, sauerkraut, fermented fish and sausages. They can be considered as natural biological preservatives in fermented foods and inhibit harmful bacteria.
Lactobacillus sp. Not only has the effect of preserving food products but also has good effects on the health of humans and animals, many studies have proven that Lactobacillus strains have the ability to pass through the stomach to colonize the intestinal tract. human and animal intestines promote host health (Slover and Danziger, 2008).
The defense mechanism of the host intestinal system of lactic acid bacteria is due to the ability of Lactobacillus to: stimulate immunoglobulin production, induce interferon, macrophages, acidify the intestinal environment, reduce blood cholesterol, and produce compounds secondary substances, bacteriocin production and the ability to occupy the mucus membrane of the intestines to inhibit or destroy pathogenic bacteria, making the intestinal system healthier, helping to increase the health of the host. (Sanders, 1993; Schiffrin and Blum, 2001).
Due to the role of Lactobacillus sp. increasingly being discovered and applied more widely in the field of food processing, pharmaceuticals and aquatic veterinary drugs, so the demand for consumption in the world is increasing. However, the production efficiency and survival rate of Lactobacillus sp. in the storage process is usually not high, so the production cost is high and the preservation time is short. Many studies have shown that the viability of Lactobacillus plantarum after drying is 0.85% (Lapsiri et al., 2012). Meanwhile, the survival rate of L.acidophilus after drying was 2.5% (Behboudi-Jobbehdar et al., 2013). With low survival rates after drying, short storage time, it will push the cost of probiotics up very high and bacteria damage a lot, so if the storage time is prolonged, the density will not be enough to determine the effectiveness of probiotics. on test subjects.
This, in turn, has a major impact on the widespread use of Lactobacillus, especially in aquaculture (Walker et al., 1999). Therefore, in recent years, there are many studies aimed at improving the survival of Lactobacillus sp. during the production and storage of Lactobacillus sp. Many studies have demonstrated that using external environmental stress such as temperature, pH, osmotic pressure, oxygen, high pressure, and lack of nutrients, bacterial cells will react to produce protective compounds. It is these compounds that will help bacteria increase the survival rate in the process of drying and preserving biomass. In addition, when bacteria were cultured under stressful conditions, a large change in the proteomic system was also recorded (Rallu et al., 1996; Sanders et al., 1999; Van de Guchte et al., 2002). ; Champomier-Verges et al., 2002).
Heat stress
Increasing or decreasing the culture medium temperature below the lethal temperature for a certain period of time before subjecting the bacterial cell to the lethal temperature threshold will increase the viability of the bacteria when the cell is subjected to the lethal temperature. stored at lethal temperatures. The genus Lactobacillus delbrueckii sub sp.Bulgaricus is very sensitive to temperature, if these bacterial cells are treated in the exponential phase at 650C for 10 minutes, the density of bacteria after treatment will be 0.0001–0.015% (Gouesbet) et al. 2001). But if stressing the culture of these bacteria was carried out at 500C for 30 minutes before subjecting them to lethal temperature (650C for 10 minutes), the survival rate increased from 10-1000 times depending on the condition. each strain. Many other studies have also demonstrated a positive effect on the survival rate of Lactobacillus acidophilus NCFM, Lactobacillus casei LC301, Lactobacillus helveticus LH212 (Broadbent et al., 1997), Lactobacillus collinoides (Laplace et al., 1999). , Lactobacillus paracasei NFBC 338 (Desmond, 2005) and Lactobacillus plantarum DPC2102 (Jordan and Cogan, 1999) when cells were subjected to moderate heat treatment before exposing them to a lethal temperature threshold.
In general, environmental stress can be divided into two groups:
Group 1 is the group that uses physical and chemical factors to stress bacteria during the culture process in order to increase the bacteria’s resistance to the impact of the same physical or chemical factors, but with the same intensity or intensity. Higher levels could thus improve the viability of the bacteria during production and storage (Sanders et al., 1999).
Group 2 is the use of an adverse environmental agent that helps the bacteria respond to more than one agent (Hecker et al., 1996; Pichereau et al., 2000). If pretreatment of L.acidophilus with NaCl during biomass culture will help them enhance resistance to bile salts and higher temperature than the control group (Kim et al., 2001). For bacteria L. paracasei NFBC 338, if they were pretreated with 0.3 M NaCl solution, the survival rate when treated at temperature (600C for 30 minutes) was (33.5% versus 0.1% in the control group) (Desmond et al., 2002; Desmond, 2005) If the NaCl concentration was increased to 0.4 M NaCl, it was still effective against all Lactobacillus sp., but replacing NaCl with 0.66 M trehalose also resulted However, this result is not true if sucrose is used at a concentration of 0.48 M (Gouesbet et al., 2001). According to Desmond et al. (2001), Desmond et al. (2002) and Desmond (2005) the heat tolerance of probiotic bacteria L. paracasei NFBC 338 was increased by pretreatment with NaCl while pretreatment with bile salts or H2O2 did not increase the heat resistance of this bacteria.
The survival rate of Lactobacillus sp. after heat stress at different thresholds outperformed those without heat stress in the face of lethal conditions. The survival rate increased from 5 to 700 times compared with the control. The results are presented in Table 1.
Table 1: Increase in survival rate of Lactobacillus sp. under heat stress before being treated in lethal conditions
Deadly Epilepsy Survival Rate Sub-lethal stress Survival rate increase
L.helveticus LH212 630C, 20 min 0.1–1 520C, 20 min 11
L.acidophilus NCFM 630C, 20 min 0.1–1 500C, 20 min 27
L. acidophilus LA1–1 600C, 30 min 0.003 530C, 30 min 166
L. casei LC301 540C, 20 min 0.1–1 420C, 20 min 5
L. paracasei NFBC338 600C, 10 min ND 520C, 15 min 300–700
L. collinoides 520C, 30 min 0.48 420C, 90 min 24
Adaptive training in low temperature
In addition to heat stress, training for low temperature adaptation is of great importance because many fermentations with Lactobacillus sp. The finish is usually preserved by low temperature or freeze-dried. If the bacteria can adapt, the viability will increase during processing and storage. Furthermore, the high viability of Lactobacilli during low-temperature storage of fermented products and probiotics before consumption is a decisive factor for the efficacy and effectiveness of Lactobacillus sp.
Lactobacillus can naturally adapt to a decrease in temperature. They continued to grow but at a reduced growth rate after the temperature dropped about 200C below the optimum temperature. Lactobacillus sanfranciscensis, L. plantarum, L. brevis, L. hilgardii, L. alimentarius and L. fructivorans grow optimally between 30 and 370C. They can grow at 150C but not below 70C. At 150C, L. sanfranciscensis CB1 has a lag phase of about 2 to 5 hours and is the lowest about 10 hours compared to the optimal culture temperature. In contrast, when the temperature is rapidly reduced from 30 or 370 C to 150 C, the growth of cells in the middle of the growth phase may temporarily stop.
But if stress acclimation is applied for about 30 to 120 minutes before allowing them to grow again. After stress acclimation, the generation time for L. sanfranciscensis CB1 is about 3 hours (De Angelis et al., 2004; De Angelis and Gobbetti, 2004).
Freezing can cause damage to cells by the formation of ice crystals but also by osmolarity due to the high concentration of intracellular solutes. Membrane damage and denaturation of macromolecules are determinants of survival after freezing (Franks, 1995; Thammavongs et al., 1996). However, other factors such as strain and species differences, growth conditions, age of bacteria, and nature of the flux also influence the persistence of Lactobacilli after freezing (Champagne et al. al., 1991; Baati et al., 2000).
Survival after freezing of L. plantarum DB200, L. brevis H12, L. plantarum 20B and L. sanfranciscensis CB1 cultured at 300C was 1.0, respectively; 0.25; 0.12 and 0.04% (De Angelis and Gobbetti, 2004).
When bacterial cells were subjected to freezing at 150C for 2 h before freezing, the viability was increased approximately 10-fold for L. sanfranciscensis CB1, 25-fold for L. plantarum DB200 and L. brevis H12 and 100 times higher for L. plantarum 20B (Table 2).
Table 2: Increase in survival rate of Lactobacillus sp. under heat stress before being treated in lethal conditions.
Deadly Epilepsy Survival Rate (%) Sub-lethal stress Survival rate (%)
L. plantarum 20B 4 freezing cycles 0.12 150C, 2 hours 11.2
L. plantarum ATCC14917 4 freezing cycles 1 150C, 2 hours 25
L. sanfranciscensis CB1 4 freezing cycles 0.04 150C, 2 hours 0.4
L. brevis 12 4 freezing cycles 0.25 150C, 2 hours 5.8
L. acidophilus 4 freezing cycles 40.0 220C, 6 hours 85
L. delbrueckii ssp. Bulgaricus
CIP101027T 4 freezing cycles 6 280C, 24 hours 25
L. lactis MG1363 4 freezing cycles 0.1 10 0C, 4 hours 10
L. lactis NZ9000 4 freezing cycles 5 10 0C, 4 hours 70
Surviving in a highly acidic environment is a positive effect of the bacteria’s adaptation to a low pH environment. This adaptive process is known as the acid concentration response (ATR) mechanism (Foster and Hall, 1991). Bacterial cells were acclimatized to a low pH medium (30 min and pH 4.75). Bacteria L. subbrueckii sub sp. Bulgaricus was able to increase tolerance by about 250 times compared to the control when tolerating at lethal acid concentrations (30 min at pH 3.6) compared with the control.
Incubation of L. colinoides at pH 3.5 for 30 minutes resulted in 0.015% of viable cells. However, when the cells were acclimatized to the medium with pH = 5.0 for 90 minutes, the viability of bacteria increased 1600 times compared to the control. The density of L. sanfranciscensis CB1 decreased rapidly when changing from pH 6.4 to pH 3.2–3.4. However, if the bacteria were stimulated by stress at pH 5.0 for 1 h before treatment at pH 3.2–3.4 for 10 h, the viability increased 46103-fold (De Angelis et al., 2001).
Lactobacilli are often exposed to changes in solute concentrations in their natural habitat. However, their cytoplasmic solute concentrations need to be relatively constant (Poolman and Glaasker, 1998). A sudden increase in the osmotic concentration of the medium leads to the movement of water from the cell to the outside causing dehydration, a change in the intracellular solute concentration, and a change in the cell volume. When cells were exposed to 18% NaCl for 2 h, the survival of L. acidophilus cells was reduced to 46%. Treatment of bacterial cells for 1 h at NaCl concentration (2%) increased survival and increased resistance to bile salts (Lemay et al., 2000). Supplementation of NaCl in the presence of glycine, betaine significantly improved the survival of L. acidophilus, L. jonsonii and other Lactobacillus bacteria during lyophilization (Zink et al., 2000).
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