Probiotics are live microorganisms that can exert beneficial influences on the host animal such as maintaining the balance in intestinal microorganisms (Anee et al., 2021). Animal probiotics that are widely utilized in general include Lactobacillus spp., Bacillus spp., Aspergillus spp., Streptomyces spp., and Saccharomyces spp., and are characterized by production and secretion of various antimicrobials, organic acids, alcohols, and digestive enzymes that metabolize proteins, fats, and carbohydrates (Indira et al., 2019; Chaudhary et al., 2022). Therefore, long-term storage of probiotics is necessary and important to maintain the quality and viability of probiotics and to ensure a stable supply.

Probiotics are mainly stored via a frozen-storage method at -20–80°C after addition of cryoprotectants such as glycerol (Prakash et al., 2020; Mahmoodian et al., 2024). Glycerol at concentrations of 2–55% is a cryoprotectant that is most frequently used in microorganism preservation (Whaley et al., 2021; Murray and Gibson, 2022). Viable cell counts are diminished during frozen-storage and freeze-drying procedures and in order to prevent this, divergent cryoprotectants such as glycerol, skim milk, glucose, lactose, mannitol, sorbitol, dextran, and polyglycol are added (Romyasamit et al., 2022). Glycerol helps to stabilize frozen bacterial cells by protecting their cell membranes, which keeps the cells alive. Bacterial stocks with glycerol can be reliably stored at -80°C for many years and at -20°C for several months (Jaiswal and Vagga, 2022). Skimmed milk acts as an effective cryoprotectant through a number of key properties. It forms a protective protein layer around bacterial cells, provides essential nutrients and prevents the formation of ice crystals. It is also widely applicable and non-toxic, making it ideal for long-term storage of bacterial cultures (Ibrahim et al., 2023; Li et al., 2023). The addition of sugars protects bacteria during freezing by limiting ice crystal growth, strengthening cell membranes, balancing osmotic pressure and minimizing freeze damage (Oluwatosin et al., 2022). The effects of cryoprotectants vary depending on the types of microorganisms, composition of the culture media, and the freezing (Wang et al., 2019).

Freeze-drying is a common process for storage of microbes and starter culture preparation in feed and food fermentations. However, freeze-drying process is accompanied by a decline in cell viability. Accordingly, it is important to select a suitable cryoprotectant of various microbes. Sugars such as sucrose and trehalose have been used to preserve lyophilized bacteria, forming a glass-like structure that protects bacterial cells from ice-related damage and helps maintain their stability (Cui et al., 2022). Jawan et al. (2022) reported that A 10% (w/v) mixture of galactose and trehalose at -30°C is the optimal composition and temperature for achieving high cell viability and stability of freeze-dried Lactococcus lactis Gh1. Di et al. (2023) observed that the combination of 6% sucrose, 8% skim milk, and 4% sodium glutamate had a high viability and protective effect on the freeze-dried Streptococcus thermophilus 937, Streptococcus thermophilus Grx02, and Enterococcus faecium 218. Many studies have been conducted on the stabilization during freeze-drying procedures, but it is difficult to obtain consistent results regarding the practical applications, because various factors including strain, composition of culture solution, pH, culture time, freeze-drying methods, and types of cryoprotectants affect the bacterial stabilization (Oluwatosin et al., 2022). Skim milk is widely used as a cryoprotectant in the probiotics industry. Also, the application of a various chemical compounds for synergistic effects of cryoprotectants has been tested (Di et al., 2023). Lim et al. (2001) reported a decrease in the survival rate when Lactobacillus plantarum MG208 was treated with 10% skim milk + 1% lactose compared to 10% skim milk, and the authors mentioned that the survival rate decreased as the concentration of lactose increased. This implies that the survival rate of Lactobacillus is dependent on species characteristics and the composition of cryoprotectants.

Therefore, it is necessary to determine a suitable cryoprotectant and its appropriate concentration for the most widely utilized animal probiotics, during -20°C frozen-storage and -80°C storage after freeze-drying. This study investigated and optimized the composition of cryoprotectants that increase survivability of probiotics for storage of L. plantarum, B. subtilis, and S. cerevisiae.

Strains, media, and survival rate

Lactobacillus plantarum BBG-L30 (KACC91952P), B. subtilis BBG-B20, and S. cerevisiae BBG-Y6 strains isolated from chicken cecum, traditional fermented soybean paste, and fermented water parsley, respectively, were used in this study, which are animal probiotics retained by Bigbiogen Co., Ltd. L. plantarum BBG-L30 and B. subtilis BBG-B20 were incubated in MRS and LB media at 35°C for 24 h, respectively, and S. cerevisiae BBG-Y6 was cultured in PDB media at 30°C for 24 h. As for microbes survival rate measurements, the bacterial culture was gradually diluted with sterilized saline solution and was placed on 2% agar-containing plate media to measure CFU (colony forming units). Also, the bacillus sporulation rate was measured using an optical microscopy (Olympus BX53F). The media (MRS, LB, and PDB media), casein, skim milk, soy peptone, and tryptone were purchased from Difco. Several starches (corn starch, soluble starch) and various sugars such as arabinose, cellobiose, fructose, galactose, glucose, lactose, maltose, mannose, sucrose, and trehalose were purchased from Daejung and Sigma Aldrich, respectively.

Survival rate investigation during -20°C frozen-storage

As the cryoprotectants to be tested, 30% of arabinose, dextrin, fructose, galactose, glycerol, glucose, lactose, mannose, mineral oil, soluble starch, and sucrose were added to culture solution of each microorganism. Carboxymethyl cellulose was added at 5% due to low water solubility. The mixture was then stored frozen in a -20°C freezer for 30 days, and changes in viable cell counts were measured and expressed as survival rate (%). Sterilized distilled water was used instead of the cryoprotectants for the control group. Glycerol was added at concentrations of 10%, 15%, 20%, 25%, 30%, and 40% to examine the preservation effect during freezing for 30 days. In order to test the synergistic effect of cryoprotectant mixtures, glycerol and mineral oil were mixed at the ratio of 1:1 in L. plantarum BBG-L30 and B. subtilis BBG-B20 cultures. In S. cerevisiae BBG-Y6 culture, glycerol and trehalose were mixed at the ratio of 1:1. Then, cryoprotectant mixtures were added at concentrations of 10%, 15%, 20%, 25%, 30%, and 40% to examine the synergistic effects on survivability during freezing for 30 days.

Survival rate in -80°C lyophilized samples

Cryoprotectants (skim milk, soy peptone, tryptone, soluble starch, casein, soybean flour, tryptone), at concentrations of 0.5, 1.0, and 1.5 g, were added to 0.5 ml of each microorganism culture solution, and sterilized distilled water (4.0, 3.5, or 3.0 ml), was added to bring the final concentration of cryoprotectants to 10%, 20%, and 30% followed by drying in a freeze dryer (Ilshin BioBase) at -80°C for 24 h. In order to investigate the synergistic effect of various sugar addition to the cryoprotectants, 20%, 10%, and 10% of soy peptone were added to L. plantarum BBG-L30, B. subtilis BBG-B20, and S. cerevisiae BBG-Y6 culture solution, respectively. For assessment of a synergistic effect on survivability, several starches and various sugars such as arabinose, cellobiose, fructose, galactose, glucose, lactose, maltose, mannose, sucrose, and trehalose were added at 1% and 5%. The sugar-containing cryoprotectants were then frozen at -80°C for 24 h followed by 24 h freeze-drying. Freeze-dried microbes were diluted after grinding and were subjected to survival rate measurements.

Statistical analysis

All experiments were independently performed and results are expressed as mean ± standard deviation.

Effects of different cryoprotectants during storage at -20°C

In order to identify proper cryoprotectants for L. plantarum, B. subtilis, and S. cerevisiae, which are mainly used as animal probiotics, 13 types of cryoprotectants were treated at different concentrations, stored at -20°C for 30 days, and followed by survival rate comparison (Fig. 1). Lactobacillus plantarum with no cryoprotectants manifested an extremely low survival rate of 0.0002%, while addition of glycerol 30% resulted in a survival rate of 12.5%, which is higher compared to mineral oil with known cryoprotective preservation effects and other additives. Bacillus subtilis showed a survival rate of 0.83% in the control group, whereas addition of 30% galactose, glycerol, and mineral oil showed a survival rate of 8.33%, which is higher than that of other additives with known preservation properties. As for S. cerevisiae, the control group showed very low survival rate of 0.001%, while addition of 30% glycerol resulted in a survival rate of 25%, which is higher than the known freezing preservative trehalose or other additives. Although the addition of 30% trehalose resulted in a relatively high survival rate for S. cerevisiae, it was not effective for L. plantarum and B. subtilis. Meanwhile, mineral oil exerted some cryopreservative effects on all strains, but these effects were all lower compared to glycerol.

Fig. 1. Effect of different cryoprotectants on the survival rate of probiotics after 30 days of frozen storage at -20°C. (●) L. plantarum, (▼) B. subtilis, (○) S. cerevisiae. 1, control; 2, arabinose 30%; 3, carboxymethyl cellulose 5%; 4, dextrin 30%; 5, fructose 30%; 6, galactose 30%; 7, glycerol 30%; 8, glucose 30%; 9, lactose 30%; 10, mannose 30%; 11, mineral oil 30%; 12, soluble starch 30%; 13, sucrose 30%; 14, trehalose 30%.

Cryopreservation effects according to glycerol concentration at -20°C

In order to investigate the appropriate cryopreservant concentrations, glycerol was added at various concentrations of 10–40%, and changes in the survival rate for each probiotic were observed after one day and 30 days of freezing at -20°C (Fig. 2). In L. plantarum after 1 day of freezing, the control group showed a survival rate of 35.2%, while 10% and 15% glycerol addition manifested a high survival rate at 55.2% for both concentrations (Fig. 2A). Bacillus subtilis also showed a high survival rate at 95.2% for the control group, and 10% glycerol addition slightly improved the survival rate to 97.6%, but did not show a significant concentration-dependent effect (Fig. 2B). While the control group of S. cerevisiae showed a survival rate of 10%, the addition of 30% glycerol resulted in the highest survival rate of 83% (Fig. 2C).

Fig. 2. Effect of glycerol concentration on probiotics survival after 1 and 30 days of frozen storage at -20°C. (A) L. plantarum, (B) B. subtilis, (C) S. cerevisiae.

After 30 days at -20°C, the control group of L. plantarum had a survival rate of 0.2%, while 25% glycerol addition induced the highest survival rate of 15.8% (Fig. 2A). Bacillus subtilis showed a survival rate of 23.8%, whereas 15% glycerol addition resulted in the highest survival rate of 30.95% (Fig. 2B). Saccharomyces cerevisiae manifested a survival rate of 0.03% in the control group, whereas 30% glycerol addition gave the highest survival rate of 58.3% (Fig. 2C).

Effects of glycerol and mineral oil mixture on the survival rate of B. subtilis at -20°C

Synergistic effects of freezing preservation agents have been investigated based on the results of Fig. 1 by mixing mineral oil with the superior cryopreservative glycerol to further improve the survival rates of L. plantarum and B. subtilis, and by mixing of trehalose with glycerol for S. cerevisiae. No synergistic freezing preservation effects were observed for L. plantarum and S. cerevisiae (data not shown). However, when glycerol and mineral oil were mixed at 1:1 and added at each concentration, synergistic effects on B. subtilis were observed in all treatment groups with the mixture compared to that of glycerol only (Table 1). Compared to the survival rate (23.80%) of the non-treatment group and that (30.95%) of 15% glycerol only, the 1:1 glycerol-mineral oil mixture showed the highest survival rate of 38.1%.

Table 1

Effect of cryoprotection by glycerol:mineral oil concentration on the survival rate of B. subtilis

Concentration of cryoprotectant agent (%)	Survival rate (%) after 30 days
	Glycerol only	1:1 mixture of glycerol and mineral oil
0	23.80 ± 2.05	23.80 ± 2.05
10	23.80 ± 1.86	35.71 ± 0.26
15	30.95 ± 0.27	38.10 ± 0.89
20	28.57 ± 1.07	30.95 ± 0.54
25	23.80 ± 1.35	33.33 ± 0.20
30	26.19 ± 0.56	30.95 ± 0.44
40	22.50 ± 0.56	28.57 ± 1.52

Effects of different cryoprotectants on lyophilized samples

To prepare lyophilized samples for preservation of L. plantarum, B. subtilis and S. cerevisiae, 6 types of cryoprotectants (casein, soy peptone, skim milk, soluble starch, soybean flour, and tryptone) were added at each concentration to compare the survival rates (Fig. 3). Glycerol was found to be an effective cryoprotectant at -20°C, as shown in Fig. 1, but it is not suitable for lyophilized bacteria due to its high viscosity and hygroscopicity.

Fig. 3. Effect of different cryoprotectants on the survival rate of probiotics after lyophilization at -80°C. (●) L. plantarum, (▼) B. subtilis, (○) S. cerevisiae. 1, control; 2, casein 10%; 3, casein 20%; 4, soy peptone 10%; 5, soy peptone 20%; 6, soy peptone 30%; 7, skim milk 10%; 8, skim milk 20%; 9, skim milk 30%; 10, soluble starch 10%; 11, soluble starch 20%; 12, soluble starch 30%; 13, soybean flour 10%; 14, soybean flour 20%; 15, tryptone 10%; 16, tryptone 20%; 17, tryptone 30%.

Although the effect of cryoprotectants differed for the different probiotics, the 6 types of cryoprotectants showed higher survival rates for L. plantarum, B. subtilis, and S. cerevisiae at all concentrations compared to the non-treatment groups. The addition of 10% soy peptone resulted in the highest survival rate of 65–85% for the three probiotics. While the L. plantarum control group had a survival rate of 0.1%, the 20% soy peptone treatment had the highest survival rate at 76.8%. The addition of 10–30% of commonly used skim milk resulted in a survival rate of 20.3–21.7%. The control group of B. subtilis had the survival rate of 3.3%, whereas the addition of 10% soy peptone resulted in the highest survival rate of 83.3%. This indicates a higher freezing preservation effect compared to the survival rate of 33.30–66.70% for 10–30% skim milk addition. The control group of S. cerevisiae showed a survival rate of 1.3%, whereas the addition of 10% soy peptone resulted in the highest survival rate at 64.1%.

Synergistic effects of cryoprotectants by adding sugar to soy peptone on lyophilized samples

Sugars such as glucose, lactose, and trehalose were added to soy peptone, which has the highest freeze-drying preservation effect, to compare and increase the survival rate of the probiotics (Fig. 4). When 13 types of sugars were added to L. plantarum, the control group and the addition of 20% soy peptone showed a survival rate of 1.2% and 40%, respectively, whereas 20% soy peptone + 1% lactose significantly improved the survival rate to 80%, which was the highest survival rate of all treatments. B. subtilis showed a survival rate of 7.8% for the control group and 78.6% for the addition of 10% soy peptone, but the survival rates of both 10% soy peptone + 1% fructose and 10% soy peptone + 5% lactose were improved to 92.9%. While there was a decrease in survival with an increasing fructose concentration, there was an increase in survival with increasing lactose concentration. Compared with the survival rates of 7% for the control group and 30% for the addition of 10% soy peptone in S. cerevisiae, the addition of 10% soy peptone + 1% mannose and 10% soy peptone + 1% glucose showed improved survival rates of 52.2% and 51.3%, respectively, which were higher survival rates than those observed for the other treatments.

Fig. 4. Synergistic effect of soy peptone and other cryoprotectants on probiotic survival after lyophilization at -80°C. (●) L. plantarum (soy peptone 20%), (▼) B. subtilis (soy peptone 10%), (○) S. cerevisiae (soy peptone 10%). Numbers 3–28 contained another cryoprotectant in addition to soy peptone. 1, control; 2, soy peptone only (L. plantarum, B. subtilis, and S. cerevisiae contained 20%, 10%, 10% of soy peptone, respectively); 3, arabinose 1%; 4, arabinose 5%; 5, cellobiose 1%; 6, cellobiose 5%; 7, corn starch 1%; 8, corn starch 5%; 9, dextrin 1%; 10, dextrin 5%; 11, fructose 1%; 12, fructose 5%; 13, galactose 1%; 14, galactose 5%; 15, glucose 1%; 16, glucose 5%; 17, lactose 1%; 18, lactose 5%; 19, maltose 1%; 20, maltose 5%; 21, mannose 1%; 22, mannose 5%; 23, soluble starch 1%; 24, soluble starch 5%; 25, sucrose 1%; 26, sucrose 5%; 27, trehalose 1%; 28, trehalose 5%.

The effects of addition of diverse cryoprotectants were investigated in order to improve survivability of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae, which are used as major probiotics, during frozen-storage at -20°C. In the comparison of different types of cryoprotectants added in this study, glycerol addition showed the highest preservation effects for all three species (L. plantarum, B. subtilis, and S. cerevisiae) tested, compared to the other additives. For the B. subtilis strain, the addition of galactose and mineral oil yielded freezing preservation effects that were as high as glycerol, indicating a different pattern from the other strains.

The survival rate of bacteria during extended preservation decreases over time (Prakash et al., 2020). The rapid decrease of the survival rate upon storage at -20°C indicates that this condition is not appropriate for long-term storage of probiotics (Tyutkov et al., 2022). Moreover, as the treatment group with a high survival rate at 1 day after freezing at -20°C showed a decreased survival rate after 30 days, it is implied that cryoprotectant experiments require a minimum of 30 days or more. In the storage of L. plantarum, B. subtilis, and S. cerevisiae, glycerol-treated groups showed higher survival rates compared to the groups without glycerol. As a result, it is recommended that glycerol as cryoprotectant in L. plantarum, B. subtilis, and S. cerevisiae is added at 25%, 15%, and 30% concentration, respectively.

Freezing preservation effects at -80°C were observed when L. plantarum was frozen with 20% soy peptone and B. subtilis and S. cerevisiae were treated with 10% soy peptone. Overall, soy peptone exhibited high preservation effects for all 3 probiotics, and this may be explained by the buffering capacity of the protein hydrolysates, glycine, glutamic acid, and amino acid mixture in soy peptone exhibiting an influence on the preservation during freeze-drying (Shin et al., 2023).

As shown in Figs. 3 and 4, the survival rate of the probiotics was in an order of B. subtilis > L. plantarum > S. cerevisiae. This may be because the extent of damage on the yeast cells during freeze-drying procedures is greater than that of bacteria (Polo et al., 2017) and the highest survival rate of Bacillus is believed to be due to spore formations. As shown in Fig. 4, the same sugar added showed different freezing preservation effects among the probiotics. Sugars added indicated lower or higher preservation effects compared with soy peptone only. This could be explained by the different cryoprotectants composition according to the structures and characteristics of the microorganisms (Wang et al., 2019).

Taken altogether, these results indicate a suitable cryoprotectant and its appropriate concentration for L. plantarum, B. subtilis, and S. cerevisiae, which are the most widely utilized as animal probiotics, during -20°C frozen-storage and -80°C storage after freeze-drying (Table 2). During –20°C frozen-storage, B. subtilis showed higher survivability when a mixture of glycerol and mineral oil was added as cryoprotectant. During -80°C freeze-drying storage, addition of a small amount of sugar such as lactose and maltose to soy peptone allowed the maintenance of a high viability. Shin et al. (2023) reported that soy peptone combined with trehalose and maltodextrin was observed to have the highest cell survivability during freeze-drying after microencapsulation of L. plantarum. In addition, Kaewiad and Kaewnopparat (2023) determined that soybean powder, skim milk and galactooligosaccharide improved the survival rates (87.06–88.52%) of L. fermentum SK54 during freeze-drying. In conclusion, it should be important to determine optimal concentration of cryoprotectants such as glycerol and soy peptone relative to sugar based on the types of probiotics.

Table 2

Survival rate on recommended cryoprotectant agent according to animal probiotics

Storage temp./condition	Probiotics
	L. plantarum		B. subtilis		S. cerevisiae
-20°C/liquid	Controla	Glycerol 25%	Control	Glycerol:mineral oil (1:1) 15%	Control	Glycerol 30%
	0.2	15.8	23.8	38.1	0.03	58.3
-80°C/freeze-drying	Control	Soy peptone 20% + lactose 1%	Control	Soy peptone 10% + lactose 5%	Control	Soy peptone 10% + mannose 1%
	1.2	80	7.8	92.9	7.0	52.2

^aNo addition of cryoprotectant agent.

가금 맹장에서 분리한 세 가지 프로바이오틱스 Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae를 다양한 냉동보호제 조합으로 냉동 보관과 동결 건조 후 생존력을 향상시키기 위해 본 연구를 수행하였다. -20°C에서 냉동 보관 시, 글리세롤은 다른 냉동 보호제보다 프로바이오틱스의 생존율(15.8~58.3%)이 더 높았다. 동결 방지제의 효과는 보관 기간이 30일 증가함으로 따라 감소했다. 글리세롤과 미네랄 오일을 1:1로 혼합하여 보존한 Bacillus subtilis는 글리세롤 단독에(30.95%) 비해 38.1%의 더 높은 생존율로 동결방지제의 시너지 효과를 입증했다. -80°C 동결 건조동안 냉동보호제 조합에 따른 프로바이오틱스의 생존율에서 L. plantarum에 대한 동결 방지제가 20% 대두 펩톤, 1% 유당일때, 생존율이 80%으로 효과적인 것으로 밝혀졌다. Bacillus subtilis에서는 10% 대두 펩톤, 5% 유당이 92.9%의 높은 생존율로 나타났으며, S. cerevisiase에서는 10% 대두 펩톤, 1% 만노스에서 52.2%의 생존율을 보였다. 결론적으로, 저장기간동안 프로바이오틱스의 생존성을 최대화하기 위한 동결방지제 글리세롤, 대두 펩톤과 설탕의 이상적인 비율과 방법을 제시하고자 한다.

None.

The authors have no conflict of interest to report.