Safety evaluation of Lactococcus lactis IDCC 2301 isolated from homemade cheese

Abstract For applications of microorganisms as probiotics in the food industry, safety evaluation has increasingly become important to ensure the health of consumers. Although people have been using various lactic acid bacteria for different purposes, some studies have reported that certain lactic acid bacteria exhibit properties of virulence and produce toxic compounds. Thus, it is necessary to examine the characteristics associated with lactic acid bacteria that are safe for use as probiotics. This research aimed to assess the safety of Lactococcus lactis IDCC 2301 isolated from homemade cheese using in vitro and in vivo assays, including antibiotic resistance, hemolytic activity, toxin production, infectivity, and metabolic activity in immune‐compromised animal species. The results demonstrated that the strain was susceptible to nine antibiotics suggested by the European Food Safety Authority (EFSA). Whole‐genome analysis revealed that L. lactis IDCC 2301 neither has toxigenic genes nor harbors antibiotic resistance. Moreover, L. lactis IDCC 2301 showed neither hemolytic nor β‐glucuronidase activity. Furthermore, none of the D‐lactate and biogenic amines were produced by L. lactis IDCC 2301. Finally, it was demonstrated that there was no toxicity and mortality using single‐dose oral toxicity tests in rats. These results indicate that L. lactis IDCC 2301 can be safely used as probiotics for human consumption.

toxin production, hemolytic activities, infectivity in immunocompromised animal models, side effects in humans, and adverse outcomes in consumers, are needed to assess the characteristics of the probiotic strains to establish safety guidelines for probiotic organisms (FAO/WHO, 2002). It is already established that the isolation history and species identification of the probiotic source should be covered in safety aspects and provide evidence of phenotypic and/ or genotypic characteristics exhibiting that the culture does not gain antibiotic resistance traits, other pathogenic properties, or putative virulence factors (Salminen et al., 1998;Sanders et al., 2010;Vankerckhoven et al., 2008). Recent interests of probiotics increase due to scientific evidences concerning health benefits. Thus, the Korean market grew fast: from $140 million (USD) in 2015 to $770 million in 2019. Furthermore, interests of consumers are also escalating in terms of the safety of probiotics as a functional food. In accordance with this notion, Lactococcus lactis IDCC 2301 strain which is isolated from homemade cheese and commercially available is required to evaluate and assess the safety as a probiotic product.
In this study, L. lactis IDCC 2301 was assessed for safety measures using genotypic and phenotypic methods to fulfill the recommendation of the EFSA manual. First, in vitro tests were performed to determine the minimal inhibitory concentrations (MICs) against various antibiotics and the hemolytic and enzymatic activities of L.
lactis IDCC 2301. Whole-genome analysis was carried out to determine whether this strain has genes associated with toxigenic and antibiotic resistance and whether these genes can be transferred to commensal or pathogenic bacteria. Also, we determined carbohydrate patterns, biogenic amine production, and lactate formation.
For in vivo test, a single-dose oral toxicity test was performed in rats.

| Culture conditions of L. lactis
The L. lactis IDCC 2301 strain was isolated from homemade cheese and cultured facultative anaerobically in MRS medium (BD Difco) at 37°C. Cell growth was monitored by measuring the absorbance at 600 nm using a microplate spectrophotometer (BioTek).

| Determination of carbohydrate fermentation patterns
The carbohydrate fermentation patterns of L. lactis IDCC 2301 were investigated using an API50 CHL/CHB kit (BioMérieux) containing 49 selected carbohydrate sources according to the manufactures' manual.
Cells grown in MRS medium overnight were obtained after centrifugation and suspended in 10 mL of API 50 CHL medium. The concentration of cells was adjusted to 6.0 × 10 8 CFU/mL and then applied to cupules with different carbohydrates on an API 50 CHL test strip. The patterns of fermentation were determined for up to 48 h at 37°C. The color changes, which represent carbohydrate fermentation, were evaluated.

| Examination of antibiotic susceptibility
The susceptibility of L. lactis IDCC 2301 to various antibiotics, which are commonly used for the treatment of enterococcal infections, was evaluated. In this test, nine antibiotics were used, namely ampicillin, clindamycin, chloramphenicol, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, and vancomycin. In brief, the cells were grown overnight, and then, 1% (v/v) of cells was transferred to fresh MRS broth. When the cell density reached approximately 10 6 CFU/mL, 50 μL of culture broth and 50 μL of each antibiotic solution at various concentrations were transferred to a 96-well plate to obtain 3.3-6.6 × 10 5 CFU/mL and 0.125-1024 μg/mL, respectively.
Then, the plates were incubated at 37°C for 20 h, and the absorbance at 600 nm was measured using a microplate spectrophotometer (BioTek). Finally, MIC was determined as the lowest concentration in which cell growth was completely inhibited.

| Test of hemolysis
The cells were streaked onto sheep blood agar plates (BD Difco) and cultured at 37°C overnight. The hemolytic potential of L. lactis IDCC 2301 was determined by measuring the clear zones around the colonies the next day. Staphylococcus aureus subsp. aureus ATCC 25923 was used as a positive control for β-hemolysis. The strain was cultured in a brain heart infusion medium at 37°C overnight.

| Test for enzymatic activity
To investigate the activities of 19 enzymes in L. lactis IDCC 2301, the API ZYM Kit (BioMérieux) was used according to the manufacturer's protocols. Briefly, L. lactis IDCC 2301 cells were cultured in MRS medium at 37°C overnight. The cells were harvested using centrifugation and suspended in phosphate-buffered saline to adjust the final cell concentration to 1.8 × 10 9 CFU/mL. Then, the cells were cultured in MRS medium at 37°C for 4 h before applying them to the API ZYM strips. After the reaction of the cells with the ZYM-A and ZYM-B reagents for 5 min, the color changes, which represent enzymatic activity, were determined using the color reaction chart.

| Measurement of lactate formation
To analyze lactate production by L. lactis IDCC 2301, the supernatants were obtained from L. lactis IDCC 2301 grown in 10 mL of MRS medium at 37°C for 24 h using centrifugation at 7000 rpm at 4°C for 30 min and filtration using a 0.22μm pore size membrane. To quantify L-and D-lactate in the filtered supernatants, an assay kit (Megazyme) was used according to the manufactures' instructions.
The absorbance at 340 nm of the reactant was measured using a microplate reader (Bio-Rad).

| Acute oral toxicity of L. lactis IDCC 2301
Acute oral toxicity (AOT) test was carried out at the Korea Testing and Research Institute (KTR; Hwasun-gun). The AOT test was performed according to the guidelines of the organization for economic cooperation and development (OECD) for testing chemicals. In brief, female rats were randomly divided into four groups of three rats (two groups age 9 weeks; and others age 10 weeks) (OECD, 2008). Rats in the treatment group were orally administered 300 or 2000 mg of freedried L. lactis IDCC 2301 powder in 10 mL sterilized water per kg body weight. Then, the mortality, signs of body weight changes, and toxicity were monitored for 14 days. On the 14th day, the rats were euthanized by injecting 100 mL isoflurane and an autopsy was conducted to examine the organs.

| Carbohydrate fermentation ability of L. lactis IDCC 2301
Lactic acid bacteria can grow on a variety of monosaccharides.
Carbohydrates from 49 different sources were evaluated for fermentation patterns by L. lactis IDCC 2301, which is presented in  The nine antibiotics were ampicillin, clindamycin, chloramphenicol, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, and vancomycin. We determined the following MIC values: 64 µg/mL for kanamycin, 32 µg/mL for gentamycin and streptomycin, 8 µg/mL for chloramphenicol, 4 µg/mL for vancomycin and tetracycline, 2 µg/mL for ampicillin, and 1 µg/mL for erythromycin and clindamycin, indicating that L. lactis IDCC 2301 was susceptible to all the antibiotics tested ( Table 2). The whole-genome sequence of L. lactis IDCC 2301 showed the highest similarity based on average nucleotide identity (ANI). The total length of the L. lactis IDCC 2301 genome was approximately 2.51 Mb with a GC content of 35.14% and 2403 functional genes (CDS) (Figure 1). In agreement with the MIC tests, the whole-genome analysis of L. lactis IDCC 2301 showed that it harbored no gene or similar genes associated with antibiotic resistance in its genome (Figure 1).

| Hemolytic property and biogenic amine production
To determine the pathogenicity of L. lactis IDCC 2301, an evaluation of hemolytic properties is tested. L. lactis IDCC 2301 was found to be negative for hemolytic activity, whereas Staphylococcus aureus ATCC 25923 induced β-hemolysis on sheep blood agar as a positive control in our analysis (Table 3). Fermented foods contain a lot of biogenic compounds and they may have some biological functions, such as bioactivities that can cause a variety of adverse effects on human health (Erdag et al., 2018). In this study, we demonstrated that five types of biogenic amines, namely, cadaverine, histamine, putrescine, 2-phenethylamine, and tyramine, were not detected in L. lactis IDCC 2301.

| Enzymatic activity test
Since enzymatic activity of microbes is important factor for safety of probiotics, we tested various enzymatic activities including βglucuronidase, esterase, and lipase. In our study, L. lactis IDCC 2301 found to have no β-glucuronidase activity (Table 4). However, five enzymes showed positive activities, namely, leucine arylamidase, αchymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and β-glucosidase. However, most of the enzymes did not show any activity. Thus, L. lactis IDCC 2301 seems not to produce toxic chemicals during the fermentation.
Based on the strains and growth conditions, L-lactate is predominantly produced; however, D-lactate can also be produced from pyruvate (Zuniga et al., 1993). Because it is difficult for D-lactate to be metabolized, it can accumulate in the human body, leading to D-lactic acidosis (Petersen, 2005;Schiraldi et al., 2003). Thus, D-lactate formation by LAB is a critical factor for safety assessment. In this study, the production of lactate by L. lactis IDCC 2301 was verified to be 100% of L-lactate (Table 5).

| Single-dose acute oral toxicity of L. lactis IDCC 2301
To evaluate the safety of L. lactis IDCC 2301 in vivo, single-dose acute oral toxicity tests with four test groups were conducted for 14 days (Table 6). It was observed that a single oral dose of 0.23-1.6 × 10 12 CFU and 0.22-1.6 × 10 12 CFU of L. lactis IDCC 2301 did not cause mortality and toxicity in 9-week-old and 10-week-old rats,  respectively. Moreover, there were no significant changes in the appearances, behaviors, body weight (Table 6), and feed intake of the rats. Throughout the autopsy, no gross pathological change was observed in all rats. Therefore, it was concluded that there is no evidence of any toxicity caused by L. lactis IDCC 2301 in the rats.

| D ISCUSS I ON
Studies have shown that L. lactis have immense bioeconomic value because of its diverse applications in food industries. Although L.
lactis is considered a nonpathogenic organism, care must be taken before it is used for human consumption. Recently, Karaaslan et al., Topcu et al., and Uchida et al. reported symptoms of L. lactis infection in children (Karaaslan et al., 2016;Topçu et al. 2011;Uchida et al., 2011). Hence, efficient use of lactic acid bacteria requires a complete understanding of many aspects of bacterial physiology, such as consumption of proteins and sugars for growth, lactate formation from sugars, synthesis of flavoring substances involved in cheese, and relation in different fermentations. In this study, we investigated the safety properties of L. lactis IDCC 2301 isolated from homemade cheese using in vitro assays, and these bacteria Forty-nine selected carbohydrate sources were used, but after incubation, the strain fermented 14 carbohydrates, namely glucose, galactose, fructose, ribose, mannose, N-acetyl-glucosamine, esculin, cellobiose, maltose, sucrose, trehalose, gentiobiose, amygdaline, salicin, and amidon; moreover, amygdaline, salicin, and amidon were uti- Bacterial hemolysin is responsible for the hemolytic activity, and it induces red blood cell lysis and consequently mild to severe infection by various pathogens (Nodzo et al., 2014). Therefore, before selecting probiotics, hemolytic activity analysis is very crucial (Sorokulova et al., 2008)  Ll a , 300 9-week-old 215.2 ± 4.7 236.4 ± 9.1 243.9 ± 5.7 256.5 ± 9.5 260.5 ± 7.0 common commensal microorganisms. Observation of physical changes by hemolytic activity after culturing the selected microorganism on an animal or human blood-containing medium is commonly used to evaluate the hemolytic activity. In this study, L. lactis IDCC 2301 showed no hemolytic activity on sheep blood agar plates.
Biogenic amines, decarboxylation of amino acid products, and low molecular weight compounds are produced by lactic acid bacterial fermentation (Barbieri et al., 2019). These compounds have some negative health effects when the amine-metabolizing power of the human body deteriorates (Alvarez & Moreno-Arribas, 2014). These amines can be formed in fermented food during fermentation or decarboxylase activity by bacteria. However, the type and level of biogenic amines formed in food products depend on the composition of the food and the growth of bacteria during the processing and storage of foods. Protein-rich fermented foods contain a high amount of biogenic amines (Jeong & Lee, 2015). For food safety purposes, tyramine and histamine are considered the most important amino acids and are responsible for various poisoning in fish and food-induced migraines (Ban et al., 2020). In our study, biogenic amines, including tyramine, histamine, putrescine, 2-phenethylamine, and cadaverine, were not detected in L. lactis IDCC 2301.
From the enzymatic activity perspective, the screened L. lactis strain was determined using the semi-quantitative API ZYM system. β-Glucosidase of lactic acid bacteria can hydrolyze glucose conjugates from plants, forming a variety of secondary metabolites. These metabolites function as health-promoting substances (e.g., antioxidants) (Michlmayr et al., 2013). However, the enzymatic activity of β-glucuronidase is important; it can indicate whether the carcinogenic compounds are present or not. It has been reported that β-glucuronidase produced by microorganisms can develop toxic steroids, such as estrogen, or carcinogenic compounds, resulting in an increased risk for colorectal cancer (Kim & Jin, 2001). In this study, we confirmed L. lactis IDCC 2301 has no β-glucuronidase activity. The leucine arylamidase activity of the selected strain of L. lactis was similar to that reported by Requena et al. and Otero et al. (Otero et al., 2001;Requena et al., 1991).
However, the activities of valine and cystine arylamidase were not detected. These results are consistent with those of several studies (Otero et al., 2001;Requena et al., 1991;Tzanetakis & Litopoulou-Tzanetaki, 1989). Requena et al. and Otero et al. re-ported similar results and found that the activity of leucine arylamidase was high in some strains of L. plantarum and L. casei subsp.
casei (Otero et al., 2001;Requena et al., 1991). Considering all these results, L. lactis IDCC 2301 could be suitable probiotics for industrial application with respect to safety issues.

| CON CLUS ION
The utilization of probiotics in the food industry is gaining popularity, especially in the dairy sector. However, safety evaluation is the most important criterion before using the selected probiotic for human consumption. For safety assessment, antibiotic susceptibility, biogenic amine formation, and hemolytic activities need to be considered according to the 2002 FAO/WHO guideline. In this study, we evaluated the safety parameters of L. lactis IDCC 2301 isolated from homemade cheese. However, we did not find any antibioticresistant gene through antibiotic susceptibility testing. Moreover, no biogenic compounds were produced after 24 h of incubation with the formation of only L-lactate (18.27 ± 0.40 mg/mL). Poor hemolytic activity is considered a low risk for humans, and fortunately, in our study, the strain showed negative activity on sheep blood agar.
Depending on the antibiotic, MIC values ranged from 1 (erythromycin and clindamycin) to 64 (kanamycin). L. lactis 2301 showed some enzymatic activities, but, importantly, β-glucuronidase activity was absent. Hence, L. lactis IDCC 2301 can be used safely as a starter organism in cheese and other fermentation processes because they did not produce biogenic amines and did not show any hemolytic activity.

CO N FLI C T S O F I NTE R E S T
All authors declare that they have no competing interests.

E TH I C S A PPROVA L
Not applicable.

CO N S E NT FO R PU B LI C ATI O N
All authors read and approved the final manuscript for publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are openly available in a public repository that issues datasets with DOIs.