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Analysis of factors affecting the freezing resistance of high-density fermented lactic acid bacteria

Study on Influencing Factors of Anti-freezing of High-density Fermentation Lactic acid Bacteria YAN Tao, ZHU Jian-guo, JIANG Tian, CHEN Ke-ke, FANG Shu-guang (Jiangsu Wecare Biotechnology Co., Ltd., Jiangsu 215200, Suzhou , China)

Abstract: With the rapid development of microecological preparations, lactic acid bacteria, as a type of probiotics, have attracted more and more attention for their probiotic effects. At present, the preparation of lactic acid bacteria powder by freeze-drying is one of the main steps in the preparation process. However, the freeze-drying process will inevitably cause varying degrees of damage to the bacteria, thereby affecting the number of viable cells of the freeze-dried powder. The high-density fermentation process of lactic acid bacteria will also affect the number of viable cells of the freeze-dried powder. Analyze the effects of high-density lactic acid bacteria fermentation process and freeze-drying process on lactic acid bacteria freezing resistance. Through the comprehensive analysis of these related influencing factors, a new idea is provided for improving the frost resistance of lactic acid bacteria and then increasing the number of viable cells of the freeze-dried powder.

Keywords: Lactic acid bacteria; Probiotics; High-density fermentation; Freezing resistance; Freezing resistance mechanism

As one of the probiotics, lactic acid bacteria are widely used in health food, medicine and food industries. Vacuum freeze-drying in the production process is one of the main steps in the preparation process. The frost resistance of the lactic acid bacteria directly affects the number of live bacteria in the freeze-dried powder. The freeze-drying process and even the high-density lactic acid bacteria fermentation process conditions in the early stage will affect The freezing resistance of lactic acid bacteria [1], and the influence of fermentation process conditions on the freezing resistance of lactic acid bacteria is easily overlooked by researchers [2]. Different types of lactic acid bacteria have different anti-freezing capabilities, but the relevant mechanisms in the anti-freezing process are basically the same [3]. This article analyzes the influence of factors such as medium composition, culture temperature, pH value, protective agent, freeze-drying process on the frost resistance of lactic acid bacteria in the fermentation process, in order to improve the frost resistance of lactic acid bacteria, and then improve its freeze-dried powder activity The bacterial count level provides new ideas. 1 The influence of the fermentation process of high-density lactic acid bacteria on its freezing resistance 1.1 The influence of the composition and content of the medium on its freezing resistance The carbon and nitrogen sources in the medium components are the main influencing factors, and they can affect the bacterial cell membrane. The content of unsaturated fatty acids has an impact. Choosing a suitable carbon source can increase the content of unsaturated fatty acids in the cell membrane, and its content is directly proportional to the fluidity of the cell membrane. Therefore, it can improve the fluidity of the cell membrane and make the cell membrane more fluid. It exhibits different degrees of freeze resistance, which greatly increases the number of viable cells after freeze-drying [4-8].

In addition to the carbon and nitrogen sources in the medium will affect the unsaturated fatty acid content in the lactic acid bacteria membrane, different types of nitrogen sources and carbon sources will also change the morphology of the lactic acid bacteria and change the resistance of the bacteria to the surrounding environment. Shao Yuyu et al. [9] added yeast extract to the commonly used lactic acid bacteria (De Man, Rogosa and Sharp, MRS) culture medium, and the length of the bacteria of Lactobacillus bulgaricus ND02 was twice that of the unadded bacteria, and the surface area of ​​the bacteria increased , It will increase the mechanical damage of the ice crystals formed in the freezing process to the cell membrane of the bacterial cell, which will lead to the massive death of Lactobacillus bulgaricus. Different carbon and nitrogen will affect the metabolic pathway of lactic acid bacteria, and then produce different metabolites. The accumulation of some metabolites will also have a certain impact on the freezing resistance of lactic acid bacteria [10]. For example, Leuconostoc mesenteroides grown on a medium containing sucrose or fructose, its metabolites can produce mannitol, which can maintain the activity of lactic acid bacteria in low water conditions, and can also play an antioxidant role [ 11]; also for trehalose, when there is no quick-acting carbon source (such as glucose) in the medium, it will be used as a carbon source by lactic acid bacteria as one of the medium components, and its metabolites, such as lactic acid, will instead cause surrounding bacteria. The decrease of environmental pH value affects the ion balance inside and outside the bacteria, which in turn increases the mortality rate [12]. In addition, studies have also found that when lactic acid bacteria grow in an environment containing certain specific carbohydrates, the extracellular polysaccharides produced by their metabolism can also improve the freezing resistance of lactic acid bacteria [13]

When calcium ions are added to the culture medium, the calcium ions can neutralize the metabolites lactic acid and other acids produced by the lactic acid bacteria during the cultivation process to generate lactate calcium salt, slow down the degree of pH drop, and reduce the effect of overacid on lactic acid bacteria. Body growth affects. During the freeze-drying process, the bacterial body changes from a liquid state to an ice crystal state, and the fluidity of the bacterial cell membrane is reduced. 

The presence of calcium ions can increase the ratio of unsaturated fatty acids to saturated fatty acids, and the greater the ratio, the greater the lactic acid bacteria The stronger the anti-freezing ability [14]. In addition, studies have also found that most lactic acid bacteria are anaerobic or facultative anaerobes, which are more sensitive to oxygen free radicals, and the presence of manganese ions can bind to superoxide dismutase, stabilize the structure of the enzyme, and ensure the catalytic activity of the enzyme. , So as to improve the activity of lactic acid bacteria under adverse conditions of low temperature, and have a certain promotion effect on the antifreeze performance of lactic acid bacteria [15]. Therefore, adding appropriate metal ions to the culture medium can increase the content of unsaturated fatty acids, improve the antioxidant performance of lactic acid bacteria, and thereby enhance the freezing resistance of the bacteria. 

Adding an appropriate amount of Tween-80 can increase the content of unsaturated fatty acids in the cell membranes of lactococcus and lactobacillus cells [16], increase the fluidity of the cell membranes under low temperature conditions, and thereby improve the antifreeze performance of lactic acid bacteria. If calcium ions and Tween-80 are added to the medium at the same time, the antifreeze performance of the bacteria can be more effectively improved. The above research shows that different nitrogen sources and carbon sources in the culture medium have different effects on the antifreeze performance of different bacteria. Nitrogen sources from animal sources can improve the antifreeze resistance of the bacteria, and the use of suitable nitrogen sources and carbon sources can make the bacteria produce It can protect the antifreeze metabolites of the bacteria. Adding trehalose needs to consider the impact of acid production on the bacteria after being metabolized. Therefore, the amount of addition must be controlled within a reasonable range. In the process of changing the nutrient composition of the culture medium of lactic acid bacteria, the number of viable bacteria in the fermentation broth is the most basic reference index. At the same time, it is more important to consider that the lactic acid bacteria cultured in this medium will be used in the later vacuum freeze-drying process. The influence of freezing resistance in order to obtain an economical and practical medium formula. 1.2 The influence of the growth temperature of lactic acid bacteria on its frost resistance When lactic acid bacteria are fermented at high density, the growth temperature of lactic acid bacteria will also affect the fatty acid content of the cell membrane of the bacteria, which in turn affects the frost resistance of the lactic acid bacteria. 

Most lactic acid bacteria will increase the content of unsaturated fatty acid C 18:2 in the cell membrane when the temperature is 3 ℃ ~ 5 ℃ below the optimum temperature, and the fluidity of the cell membrane will be correspondingly enhanced, which will eventually improve its anti-freezing ability, but it needs Grasp the temperature change, it should not be too low, otherwise it is not conducive to the growth of the bacteria [17]. Low temperature will also promote the synthesis of some lactic acid bacteria polysaccharides, thereby improving their freeze-drying survival rate [18]. When Lactobacillus acidophilus RD758 and CRL640, Lactobacillus coryneformis Si3 and Lactobacillus bulgaricus L2 were fermented and cultured at 30, 35, and 37 ℃, respectively, by comparing the survival rate of freeze-drying before and after freezing, it can be seen that it is antifreeze when fermented at 30 ℃. The strongest ability, the survival rate of freeze-drying is as high as 67%, which is 30% higher than before. The extracellular polysaccharide produced by Streptococcus thermophilus cultured at 30 ℃ is 2 to 5 times higher than that at 37 ℃, and this polysaccharide The substance has a significant effect on the frost resistance of lactic acid bacteria [18]. Therefore, appropriately lowering the culture temperature can change the content of unsaturated fatty acids in the membrane, which is beneficial to improve the fluidity of the cell membrane during lyophilization; at the same time, it can also promote the polysaccharide synthesis of lactic acid bacteria and improve the freezing resistance of lactic acid bacteria. 1.3 The effect of constant pH during fermentation on the frost resistance of lactic acid bacteria The control of pH value during the fermentation process of lactic acid bacteria will also affect its frost resistance. Studies have found that different lactic acid bacteria (cocci, bacilli, etc.) have significant differences in their fermentation pH values ​​for antifreeze resistance. 

Studies have found that lactococci are more suitable for growth under fermentation culture conditions with a pH value of 6.0; while lactobacilli are suitable for a pH value of 5.0 Grow under the fermentation and culture conditions. The main reason is that different pH values ​​will increase the content of unsaturated fatty acids in the cell membrane, improve the fluidity of the cell membrane of the cell, and then improve its antifreeze performance [19]; ​​too much acid and too alkali are also not conducive to the growth of the cell. In addition, studies have also found that [20] different constant pH values ​​will affect the morphology of the bacteria. When Lactobacillus bulgaricus ND02 grows in a common MRS medium with a constant pH value from 5.7 to 5.0 during fermentation culture, its bacteria The body shape changes from a slender shape to a stubby shape, and when the shape is short and thick, the freeze-drying survival rate is better than that of the slender shape. Therefore, it is necessary to control the suitable fermentation pH value for different lactic acid bacteria. Culturing under the corresponding fermentation pH value conditions can affect the content of fatty acids in the bacterial membrane, change the morphology of the bacteria, and improve the antifreeze ability of the bacteria.

1.4 The influence of the neutralizer in the fermentation process on the frost resistance of lactic acid bacteria

During the fermentation process of lactic acid bacteria, due to the growth of its bacteria, the metabolites that affect the pH change in the medium are mainly lactic acid. Of course, there are other acids (such as acetic acid, formic acid, etc.). The excessively low pH value will affect the lactic acid bacteria. The growth produces feedback inhibition. Therefore, it is necessary to neutralize acidic substances with a neutralizer during the fermentation process to maintain a constant pH value in the culture medium and ensure the best growth environment for the bacteria. Neutralizers are generally alkaline, such as sodium carbonate, ammonia, sodium hydroxide, etc., and the use concentration (mass volume ratio) is generally 20% to 30%. Since ammonia can free NH 4 +, it easily penetrates the cell wall, stimulates the growth of bacteria, increases the number of viable cells in the fermentation broth, and then increases the number of viable cells of the lyophilized bacterial powder. It is more suitable as an acid-base neutralizer [ 21]; But when Zhang Xingchang et al. [22] studied Streptococcus thermophilus ND03, they found that using 30% sodium carbonate as a neutralizer saves 20 mL than 25% ammonia, and sodium carbonate as a neutralizer. After neutralization with acid, It will produce CO 2 and form an anaerobic environment, and most of the lactic acid bacteria are facultative and partial anaerobic growth, creating a more suitable environment for the growth of anaerobic lactic acid bacteria. In summary, the acid produced by the metabolism of lactic acid bacteria during growth reduces the pH value of the environment, thereby affecting the optimal environment for its growth. During the growth process, adding an appropriate acid-base neutralizer to maintain a constant pH value is more conducive to maintaining the best environment for the growth of lactic acid bacteria, thereby increasing the number of viable bacteria powder after freeze-drying.

1.5 The impact of the harvest time of lactic acid bacteria on its frost resistance

During the fermentation process of lactic acid bacteria, the growth of the bacteria will go through a lag phase, a logarithmic phase, a stable phase and a decay phase. Different harvest periods of bacteria have a significant impact on the number of viable bacteria. Studies have found that the survival rate of the bacteria harvested by centrifugation, emulsification, and freeze-drying after the fermentation culture reaches the initial stage of the stable phase is 8 times higher than the survival rate of the bacteria harvested in the mid-log phase [23]. The survival rates of Lactococcus lactis DRC-2 and DRC-2C in the log phase were 28.5% and 34.7%, respectively, while the survival rates in the stable phase reached 60% and 61.2%, an increase of 2 to 3 times [24]. Rault et al. [25] found that the freezing resistance of the bacteria harvested in the stable phase of Lactobacillus bulgaricus CFL1 was better than that of the log phase; Ampatzoglou et al. [26] found that they were harvested in the stable phase when studying Lactobacillus rhamnosus LGG. The frost resistance of the bacterium is better than that of the logarithmic phase; Louesdon et al. [27] studied Bifidobacterium longum RO175 and also showed that the lyophilization resistance of the bacteria harvested in the stable period is better than that of the anti-freeze drying. Several periods. The reason why the frost resistance of the bacteria harvested in the stable phase is better than that of the log phase is that the bacteria in the stable phase are due to changes in culture conditions (accumulation of low substances, changes in pH, etc.) and lack of nutrients. , So that the growth and death of the bacteria are in a state of dynamic equilibrium, thereby inducing a series of stress responses to adapt to the environment, and improving its resistance to adverse environments [28], such as its cell membrane lipids in the stable phase. The composition (increased content of cyclopropanated fatty acid (cycC 19:0)) changes, thereby enhancing the anti-freeze-drying ability of lactic acid bacteria [29-30]. In addition, during the stationary phase, the expression level of stress protein of lactic acid bacteria and the structure of the cell wall also changed drastically, which led to an increase in resistance to adverse conditions such as freeze-drying [31]. Of course, there are exceptions. For example, Yuan Jieli et al. [32] tested the anti-freeze drying survival test of Lactobacillus brucei R1102 harvested in the logarithmic phase and the stable phase, and found that there was no significant difference between the two, which may be different strains. It is caused by the difference between fermentation and culture [33]. In summary, the anti-freeze-drying performance of lactic acid bacteria in the stable growth phase is stronger than that of other growth phases, and the differences between strains must also be considered.

1.6 The effect of treatment after the completion of high-density fermentation of lactic acid bacteria on its freezing resistance

After the fermentation of lactic acid bacteria, the frost resistance can be improved by adjusting the pH value of the fermentation broth and adding specific substances. Studies have found that by adjusting the pH value of the fermentation end point of Streptococcus thermophilus ND03 from 5.8 to 6.6, the survival rate of the bacteria obtained after centrifugation, emulsification, and freeze-drying is greatly improved [34]. Zhang Zhongqing et al. [35] found that after fermentation, the fermentation broth of Lactobacillus bulgaricus ATCC11842 was added with a sterilized 2% (mass to volume ratio) sodium chloride solution, incubated at 37 ℃, 100 r/min for 2 h. After emulsifying and freeze-drying the bacteria and the protective agent, the freeze-drying survival rate reaches 65%, which is 1.43 times that of untreated. The reason is that the stimulation of sodium chloride changes the level of intracellular cofactors, increases the activity of phosphofructokinase (PFK) in the cells by 31.4%, increases the activity of enzymes, and freeze-drying can bring about lactic acid bacteria. The damage is minimized, thereby improving the lyophilized survival rate of lactic acid bacteria [36]. Therefore, after fermentation, by adjusting the fermentation pH value or adding specific substances, it is beneficial to increase the acid production activity level of the bacteria, and indirectly increase the activity of certain kinases, thereby increasing the survival rate of the bacteria during freeze-drying.

2 The effect of high-density lactic acid bacteria fermentation protective agent/lyophilization on the frost resistance of lactic acid bacteria

2.1 The effect of protective agent on the frost resistance of lactic acid bacteria

The freeze-dried protective agent has a direct impact on the survival rate of lactic acid bacteria. Therefore, the lactic acid bacteria mud is emulsified and mixed with an appropriate protective agent before freeze-drying and then lyophilized, which can improve the survival rate of lactic acid bacteria. The effect of protective agent is closely related to its chemical structure and molecular weight. In freeze-drying and storage, different substances such as sugars (sucrose, lactose and trehalose), protein compounds (skimmed milk), amino acids (glutamic acid and aspartic acid) and antioxidants (ascorbic acid) have been used as protection To improve the survival rate of lactic acid bacteria [37]. Lyophilized protective agents can be roughly divided into two types: one is small molecular weight compounds. Such as amino acids, organic acids, low-molecular sugars and sugar alcohols, etc., these small molecular substances can pass through the cell membrane of lactic acid bacteria into the cell to inhibit the formation of ice crystals, slow down the growth of ice crystals, and reduce the damage to the bacteria during freezing; The second is macromolecular substances. Such as proteins, polysaccharides, polyvinylpyrrolidone and other synthetic polymers [38]. These macromolecular substances cannot penetrate the cell membrane of lactic acid bacteria. 

Because of their strong hydrophilic properties and hydrogen bond forming ability, they are attached to the surface of the bacteria. , Forming a stable layer of water molecules, preventing the outward transfer of bound water in the cell membrane, and protecting the cell structure of the bacteria. The combination of different protective agent substance types can reduce the damage of freeze-drying to the bacteria, improve the freezing resistance of the bacteria, and then significantly improve the freeze-drying survival rate of lactic acid bacteria. Among the protective agents, carbohydrates, amino acids, Peptides and proteins are the most widely used. From the current research, carbohydrate protective agents have a significant protective effect on the freeze-drying and dehydration of lactic acid bacteria. Its mechanism of action is to inhibit the phase transition of membrane lipids, that is, when the lactic acid bacteria cells are lyophilized, sublimated, and dehydrated, the "water displacement" effect is performed, so that in the presence of carbohydrate protective agents, the state of dry membrane lipids is different from that of untreated lactic acid bacteria. 

The physiological characteristics before dehydration are similar [39], thereby stabilizing the cell structure of the bacteria and improving the survival rate of the bacteria after freeze-drying; skimmed milk contains a mixed macromolecule (whey protein and casein) and sugars, skimmed milk It can make the freeze-dried product form a porous structure, which is easier to rehydrate; after the cell membrane is dehydrated, these carbohydrate substances replace the bound water to prevent the exposure or aggregation of proteins through hydrogen bonds [40], and provide good protection for lactic acid bacteria, so it can As a protective agent for many lactic acid bacteria [41]; skimmed milk can prevent cell damage by stabilizing cell membrane components and provide a protective layer of cell protein [42-43]. The combined use of various protective agents is better than the use of them alone. It is necessary to combine the use of large molecules and small molecules in order to better exert their protective effects.

2.2 The effect of pre-freezing rate on its frost resistance

During the freeze-drying process, the freeze-drying process, such as the pre-cooling rate, will also affect the freeze-drying survival rate of the bacteria. Among them, slow freezing can increase the freezing resistance of lactic acid bacteria. The reason is that the pre-cooled lactic acid bacteria are stimulated by low temperature, which can induce the production of cold stress proteins and improve the low temperature activity of related enzymes; in addition, low temperature will make the saturated fatty acids on the cell membranes of the bacteria change into unsaturated fatty acids, increasing The fluidity of the cell membrane of the cell at low temperature. These phenomena play an important and active role in protecting the freeze-drying resistance of lactic acid bacteria in the later stage [44]. In addition, some studies have shown that when the freezing rate is between 5 ℃/min and 180 ℃/min during lyophilization, the intracellular water will completely leak out of the bacteria during lyophilization, and there will be no crystallization in the bacteria, and the survival rate of lactic acid bacteria Higher; when the freezing rate is between 180 ℃/min~5 000 ℃/min, the moisture in the bacteria is likely to form crystals during the extravasation process, which will easily cause mechanical damage to the bacteria; when the freezing rate is greater than 5 000 ℃/min At min, the intracellular water is too late to infiltrate and quickly form crystals, and the cell survival rate will be affected to a certain extent; therefore, during pre-freezing, the freezing rate needs to be adjusted according to the characteristics of different bacteria to achieve the best freezing survival rate [ 44].

3.Summary

Vacuum freeze-drying is one of the important steps in the preparation process of Lactobacillus bacteria powder. The bacteria will undergo a series of changes under cold stimulation, such as changes in cell membranes, changes in genetic material, etc. This article focuses on fermentation process, post-fermentation treatment, and protection. The influence of lactic acid bacteria on freezing resistance of lactic acid bacteria is analyzed. The composition of the medium, the temperature of the medium, the pH value of fermentation, the protective agent and the lyophilization process will all have an impact on the lyophilization performance of lactic acid bacteria. The characteristics of the strains require different control conditions, and there are also certain connections between different factors. A good grasp of the relationship between the various influencing factors is also one of the keys to improving the frost resistance of Lactobacillus. The current research on the freezing resistance of lactic acid bacteria stays at the macro level (such as culture medium, temperature, pH value, etc.), while the micro level (strain metabolism, exchange of bacteria and nutrients, strain differences, etc.) Not in-depth enough, the frost resistance of lactic acid bacteria is a very complicated process, and it still needs to be further studied in the future.

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