Preliminary purification and partial characterization of a functional bacteriocin of Lacticaseibacillus paracasei Zhang and mining for its gene cluster

doi:10.21203/rs.3.rs-3638597/v1

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Preliminary purification and partial characterization of a functional bacteriocin of Lacticaseibacillus paracasei Zhang and mining for its gene cluster

https://doi.org/10.21203/rs.3.rs-3638597/v1

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Bacteriocins produced by lactic acid bacteria have good potential for use as food biopreservatives. Lacticaseibacillus paracasei Zhang (L. paracasei Zhang) is both a food use and a probiotic bacterium. This study aimed to purify and preliminary characterize the active antibacterial metabolite of L. paracasei Zhang. The cell free supernatant of L. paracasei Zhang was collected and purified by ultrafiltration and gel filtration chromatography. The 1–3 kDa active fraction could inhibit the growth of Staphylococcus aureus but not Escherichia coli. Further antibacterial activity assays revealed its capacity in suppressing various foodborne and human opportunistic pathogens (including Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Listeria monocytogenes, and Bacillus cereus), but not fungi. The antibacterial activity showed good tolerance to heat (40℃ to 100℃), acid-base (pH 2–3 and pH 6–10), and digestions by a number of industrial and animal/human enzymes (such as trypsin, pepsin, α-amylase and protease K, except papain); these desired properties make it a suitable biopreservative to be used in harsh and complex industrial production processes. The high papain sensitivity suggested a proteinaceous/peptide nature of the bioactivity. Moreover, our genomic data mining for bacteriocin through BAGEL4 revealed an area of interest encoding a complete set of putative genes required for bacteriocin production. In conclusion, our study showed that L. paracasei Zhang can produce extracellular functional antibacterial metabolite, likely a class II bacteriocin. Our preliminary extraction and characterization of the bioactivity demonstrated that it has good potential to be used as biopreservative or agent for suppressing gastrointestinal infections.

Lacticaseibacillus paracasei Zhang

Antibacterial metabolite

Purification

Characterization

Bacteriocin

Food preservatives are often added into food products to prevent food spoilage [1]. However, with the improvement of living standards and consumers’ health awareness, chemical preservatives are increasingly rejected by consumers because of their potential toxicity or negative health effects [2]. Therefore, the development of natural lactic acid bacteria (LAB) biological preservatives with broad spectrum of antimicrobial activity, stability, and safety has attracted much attention from the food industry [3]. Biopreservatives are of potential for use in natural control of food spoilage microorganisms and foodborne pathogens. Thus, it is of interest to isolate microbes that can synthesize these active biomolecules, as well as to extract and to characterize the produced antibacterial metabolites [4].

Lactic acid bacteria are ubiquitous lactic acid-producing Gram-positive bacteria. The use of LAB in food production and fermentation not only impart unique sensory properties to the food, but also confer desirable health-promoting effects to consumers, such as promoting colonic and gut microbiota homesostasis, enhancing immunity, preventing from tumor development, and exerting anti-inflammatory and antioxidation effects [5]. In the process of growth and metabolism, LAB can secrete a variety of metabolites, such as organic acids, diacetyl, ethanol, hydrogen peroxide, exopolysaccharides, and bacteriocins, many of which show good antimicrobial effects [6, 7]. For example, it was found that L-phenyl lactic acid extracted from the fermentation supernatant of Lactiplantibacillus plantarum Z316 could inhibit the food pathogen, Salmonella enterica subsp. enterica ATCC 14028 [8]. Another study reported the inhibitory activity of some polysaccharide components in the supernatant of Pseudoalteromonas haloplanktis TAC125 against Staphylococcus epidermidis biofilm formation [9]. The bacteriocin M1-UVs300 isolated from Lactiplantibacillus plantarum plantarum M1-UVs300 exhibited a broad-spectrum antibacterial activity against both Gram- and Gram-negative bacteria [10].

Among the antimicrobial materials, bacteriocins have attracted much attention because of their enticing features, such as good safety, high efficacy in microbe suppression, non-toxicity, residue-free, low/no risk in causing drug resistance, and ease of decomposition by protease after being ingested. Bacteriocins are antibacterial peptides or precursors synthesized by ribosomes in the process bacterial growth and metabolism for enhancing survival competitiveness by suppressing surrounding microorganisms [11]; and some of which have shown strong inhibitory effect against antibiotic-resistant bacteria [12]. It was reported that most bacteria and archaea produce at least one bacteriocin [13]. Unlike synthetic drugs and chemical preservatives, bacteriocins have a rapid and powerful effect even at low doses, and they do not tend to drastically alter the natural human gut commensals [3, 14]. Their modes of action and desired inhibitory effects on potential pathogens and drug-resistant bacteria have made them a good option for use in food spoilage control and fresh-keeping [15].

Some bacteriocins have good heat, acid, and alkali resistances, but are protease-sensitive [16]. It was found that the antibacterial activity of the bacteriocin produced by Lactobacillus brucellosis retained after a harsh heat treatment (121℃ for 30 minutes), and indeed its bacteriostatic effect was even better at a higher temperature. Moreover, the bacteriocin was active within a wide pH range from pH 1–10 [17]. Bacteriocin BM1029 extracted from Lactobacillus crustorum MN047 also showed strong antibacterial activities over a wide pH range from pH 2–10 [18]. Another example is bacteriocin TF711, which was found to be active at 100℃ and in the range of pH 1–10, though it was sensitive to protease K, pepsin, and trypsin [19]. Most bacteriocins can only inhibit a specific group of microbes, such as Gram-positive bacteria; however, a small proportion of bacteriocins exhibit bacteriostatic effects on Gram-negative bacteria and fungi as well. The bacteriocins-originated from Bacillus pumilus ZED17 and DFAR8 could inhibit Gram-positive, Gram-negative bacteria, and some fungi [20]. Thus, the properties and antimicrobial spectrum of each bacteriocin seems to be unique and needs to be individually investigated.

The Lacticaseibacillus paracasei (L. paracasei) species has been widely used in the food industry, and some members of this species are known to produce antibacterial metabolites and/or bacteriocins such as paractocin SD1, bacteriocins BGSJ2-8 and paracin 54 [21–23] Some L. paracasei-originated bacteriocins have been shown to exert intestinal homeostasis and regulatory effects [24].

L. paracasei Zhang strain was isolated from a naturally fermented horse milk sample collected in Xilingol grassland, Inner Mongolia, in 2001 [25]. This strain has been shown to have good fermentation characteristics and various probiotic effects through in vitro, clinical and animal intervention trial studies. For example, it could increase antioxidation and anti-lipid peroxidation activities [26], enhance cellular/humoral immunity and tumor suppressive function [27], improve blood lipids and liver lipid metabolism [28], prevent type Ⅱ diabetes, and raise liver function and protect against liver injury [29]. Moreover, in a population trials, it has shown effective gut microbiota regulatory effects by enhancing the beneficial bacteria while suppressing the potentially harmful ones [26], and prophylactic effects against upper respiratory tract infection [30].

This study found that the genome of L. paracasei Zhang distinct bacteriocin gene cluster-encoding genomic regionss. Thus, the antibacterial activities of L. paracasei Zhang was explored by extracting, preliminarily purifying, and characterizing its extracellular antibacterial metabolite. The antimicrobial spectrum of the bioactive extract against indicator bacteria and fungi was also determined. Our results showed that L. paracasei Zhang produces a functional and stable bacteriocin with a broad antimicrobial activity spectrum, which is a novel microbial resource of potential for use as food biopreservative.

Microbial strains and cultivation

The L. paracasei Zhang was isolated from the traditional fermented horse milk (pH 3.37–3.94) in Zhenglan Banner, Xilingol, Inner Mongolia in 2001 [25]. It is preserved at the Key Laboratory of Dairy Biotechnology and Engineering of the Ministry of Education, Inner Mongolia Agricultural University. The frozen bacterial stock was activated and inoculated in liquid de Man, Rogosa and Sharpe (MRS) medium (pH 5.90) and aerobically cultured at 37℃ for 24 hours. The indicator strains of antimicrobial assay were shown in Table 1. Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), Fusobacterium nucleatum subsp. Polymorphum (F. nucleatum), Porphyromonas gingivalis (P. gingivalis), Bifidobacterium animalis subsp. Lactis Probio-M8 (Probio-M8), and Clostridium perfringens (C. perfringens) were anaerobically cultured for 12 hours, and the rest were aerobically cultured for 12h.

Table 1

List of indicator strains.
Indicator strain	Strain identification number	Cultivation conditions
Gram-negative bacterium
Actinobacillus actinomycetemcomitans	BNCC 336945	TSB, 37℃
Escherichia coli	CICC 23657	LB, 37℃
Fusobacterium nucleatum subsp. polymorphum	BNCC 336949	TSB, 37℃
Porphyromonas gingivalis	BNCC 337441	TSB, 37℃
Pseudomonas aeruginosa	ATCC 47085	LB, 37℃
Pseudomonas fluorescens	CICC 21620	LB, 30℃
Riemerella anatipestifer	-	LB, 37℃
Salmonella enterica	CICC 10982	LB, 37℃
Vibrio harveyi	CGMCC 18690	2216E, 37℃
Vibrio parahemolyticus	CICC 21617	3%Sodium Chloride Peptone Water, 37℃
Gram-positive bacterium
Bacillus cereus	CICC 10277	LB, 37℃
Bifidobacterium animalis subsp. lactis	Probio-M8	MRS, 37℃
Clostridium perfringens	CICC 22949	Cooked Meat Medium Base, 37℃
Lacticaseibacillus paracasei	Zhang	MRS, 37℃
Lacticaseibacillus rhamnosus	Probio-M9	MRS, 37℃
Lactiplantibacillus plantarum	P-8	MRS, 37℃
Listeria monocytogenes	ATCC 15313	LB, 37℃
Pediococcus acidilactici	PA-19	MRS, 30℃
Staphylococcus aureus	ATCC 12600	LB, 37℃
Streptococcus mutans	BNCC 336931	BHI, 37℃
Fungus
Aspergillus flavus	CICC 2219	PDB, 30℃
Candida albicans	CICC 32819	SDB, 30℃
Cladosporium sphaerospermum	CICC 2477	PDB, 30℃

Remarks: Trypticase Soy Broth (TSB); LB Broth (LB)༛ 2216E Liquid Medium (2216E)༛ MRS Broth (MRS)༛ Brain Heart Infusion Broth (BHI)༛ Potato Dextrose Broth (PDB)༛ Sabouraud Dextrose Broth (SDB).

Antibacterial activity assay

Antimicrobial activity was assayed by the Oxford cup method using Escherichia coli (E. coli) CICC 23657, Staphylococcus aureus (S. aureus) ATCC 12600 as the indicator strains [31]. Activated L. paracasei Zhang was inoculated in MRS medium (1%, V/V; pH 5.9), cultured for 24 hours at 37℃, and then centrifuged (4℃, 6000 × g) for 10 minutes. Cell-free supernatants (CFS) were obtained and were filtered through a 0.22 µm pore size membrane on an ultra-clean worktable. After determining the pH value of CFS, it was stored at 4℃ for use. The activated indicator bacteria were adjusted to 10⁵ CFU/mL with phosphate buffer saline, mixed with sterilized molten nutrient agar (1%, V/V), and poured (20 mL per assay) into fresh sterile plates. After the bacteria-inoculated agar solidified, Oxford cups were placed vertically on the surface of the culture medium and gently pressed to ensure good contact with the culture medium without gap. For each assay, 150 µL of L. paracasei Zhang CFS was slowly added into the Oxford cups to allow pre-diffusion of the CFS at 4℃ for 4 to 10 hours. The subsequent agar plates were incubated at 37℃ until for 12 hours for the development of inhibition zones. The diameter of the inhibition zone was measured by the linear measurement setting in a colony automatic counter. Each assay and measurement were performed in three replicates.

Fractionation of CFS by ultrafiltration

The L. paracasei Zhang CFS was desalted by using ultrafiltration membranes of molecular weight cutoffs of 1, 3, 5, and 10 kDa (GUOCHU TECHNOLOGY, XiaMen, China) until the electrical conductivity reached 3.0 ms/cm, measured by Electrodialysis equipment (GUOCHU TECHNOLOGY, XiaMen, China). The desalted CFS fractions were concentrated by spray drying (MOBILE MINOR, GEA Process engineering China Limited, ShangHai, China) with the following settings: initial material temperature, 20℃; inlet temperature, 165℃; exhaust temperature, 70℃; inlet air volume, 8.0 m³/h; air pressure, 0.3MPa; and feed speed, 8.0 r/min.

Gel filtration of 1–3 kDa CFS fractions

The ultrafiltrate fractions of 1–3 kDa were further separated and purified by gel filtration chromatography using an AKTA Avant 150 (General Electric Company, Boston, USA). Briefly, the ultrafiltrate fractions of 1–3 kDa were diluted 10 ten times with ultrapure water, and 6 mL of the diluent was loaded onto a Sephadex G25culumn (2.6×100 cm; Pharmacia Biotech, Uppsala, Sweden). Then the column was eluted with ultrapure water at a flow rate of 13 mL/min, and the eluent was monitored at optical density 280 nm (OD₂₈₀). Fractions of 30 mL were collected according to the absorbance, and 30-fold concentrated by lyophilization. Then, the high absorbance fraction or the active fraction (AF) was assayed for antibacterial activities using the Oxford cup method against 23 indicator strains or pathogens (Table 1).

Characterization of the antibacterial material in AF

Heat stability

The heat stability of AF was tested. The AF was 10-fold diluted with ultrapure water, adjusted to pH 7.0 with 5 M NaOH. The osmotic pressure of the diluted AF was measured by Automatic Cryoscopic Osmometerto (OSMOMAT 3000, Gonotec GmbH, Germany) exclude the effect of osmotic pressure on antibacterial activity. The AF was heat up for 30 minutes at different temperatures (40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃, and 121℃) before its antibacterial activities was determined by Oxford cup method. Untreated AF was used as the negative control in the assay.

pH stability

The acid-base stability of AF was tested. The same 10-fold diluted AF was adjusted to pH 2, 3, 4, 5, 6, 7, 8, 9, and 10.0 using 3 mol/L HCL or 3 mol/L NaOH, respectively. The adjusted AF samples were incubated at 4℃ for 24 hours before applying the Oxford cup method to assay changes in antibacterial activities.

Enzyme resistance

Resistance of AF antibacterial material to various enzymes was tested, including catalase (pH 7.0, S10037, YuanYe Bio-Technology, ShangHai, China), α-amylase (pH 6.5, S10003, YuanYe Bio-Technology, ShangHai, China), lysozyme (pH 6.5, RL0295, Biocare, ZhuHai, China), RNAase (pH 7.6, R1030, Solarbio Science & Technology, Beijing, China), lipase (pH 8.0, S10035, YuanYe Bio-Technology, ShangHai, China), pronase E (pH 7.0, S10014, YuanYe Bio-Technology, ShangHai, China), pepsin (pH 2.0, RM1020, RYON, ShangHai, China), trypsin (pH 7.0, RM1022, RYON, ShangHai, China), papain (pH 7.0, RM1009, RYON, ShangHai, China), and protease K (pH 8.0, RM1060, YuanYe Bio-Technology, ShangHai, China). These enzymes were dissolved in ultrapure water in the concentration of 10 mg/mL, respectively. An aliquot of 100 µL of each enzyme was added to 900 µL of ten-fold diluted AF and incubated at 37℃ for three hours, before conducting antibacterial assays by the Oxford cup method.

After the first round of assays, it was found that papain could inactivate the antibacterial activity of AF completely. Therefore, in order to further verify the effect of papain on the antibacterial activity of AF, the assay of AF resistance to papain was elaborated by incubating the AF-enzyme mix at 37℃ for 0, 0.5, 1, 3, and 6 hours, respectively. After the incubation for specific time, the enzyme was inactivated by boiling for 10 min. Afterwards, the antibacterial assay by Oxford cup method was performed.

Mining of L. paracasei Zhang genome for bacteriocin gene cluster

The whole genome sequence of L. paracasei Zhang was retrieved from the NCBI database (accession number: CP001084.2) and searched through the BAGEL4 bacteriocin online database system (http://bagel4.molgenrug.nl) to identify potential bacteriocin gene cluster.

Antibacterial activity of CFS of L. paracasei Zhang

The CFS of L. paracasei Zhang showed remarkable inhibitory effect against the indicator strain, S. aureus ATCC 12600, but not E. coli CICC 23657, indicating that L. paracasei Zhang could produce extracellular metabolites to inhibit certain pathogenic bacteria (Figs. 1A and 1B). Ultrafiltration was then performed to enrich and desalt the active metabolites, obtaining ultrafiltrates of different molecular weights (i.e., fractions < 1 kDa, 1–3 kDa, 3–5 kDa, 5–10 kDa, and > 10 kDa, respectively). The different ultrafiltrate fractions were tested for their antibacterial activity against S. aureus ATCC 12600, and the 1–3 kDa fraction exhibited the strongest activity (Table 2). In addition, in the agar antibacterial activity assay, the inhibition zone diameter of 1–3 kDa fraction against S. aureus ATCC 12600 was obviously larger than that of the original CFS (Fig. 1C), indicating the enrichment of antibacterial active metabolites. The 1–3 kDa fraction was then further purified by gel filtration chromatography.

Table 2

Anti-staphylococcus activity of ultrafiltrates of cell free supernatant of *Lacticaseibacillus paracasei* Zhang
Molecular weight range (kDa)	Anti-staphylococcus activity
<1	-
1–3	++
3–5	+
5–10	+
>10	-

Remarks: “++” and “+” represent inhibition zone size of 11–15 mm and 7–11 mm, respectively; “-” means no inhibition.

Preliminary purification of the 1–3 kDa fraction

The 1–3 kDa fraction was subjected to gel filtration chromatography. As shown in Fig. 2, the extracellular metabolites of L. paracasei Zhang were eluted as four OD_280nm peaks. Fractions from the four peaks were collected and tested for their anti-staphylococcus activity, respectively; and only peak 1 was positive. This gel filtration fraction was regarded as the active fraction (AF) and was used for subsequent experiments.

Characterization of the antibacterial activity in AF

Effect of heat on the antibacterial activity of AF

Heat treatment is a common process in food production and other industrial processes, so it would be of interest to test the heat resistance of AF [32]. The inhibition zone diameter of heat treated AF showed a decreasing trend, as the heat treatment temperature increased from 40℃ gradually to 100℃. Even after a harsh heat treatment for 30 min at 121℃, the inhibition remained obvious with a zone size reduction of only 26.15% (Fig. 3), suggesting a high heat stability of the antibacterial metabolites.

As high osmotic pressure can inhibit bacterial growth, the osmotic pressure of 10-fold-diluted AF (pH 7.0) was measured, which was about 800 mOsm/L, excluding the possibility of osmotic pressure interference on bacterial growth.

Effect of pH on the antibacterial activity of AF

Ideally, an industrial use biopreservative should have a good acid-base tolerance to ensure its stability in different matrix environments [33]. Thus, we tested the stability of the antibacterial activity of AF over a broad pH range (Fig. 4). Our results showed that it was greatly influenced by pH, with a drastic reduction in antibacterial activity against the staphylococcal indicator at pH 4 and 5 compared with a neutral pH (P<0.0001). Interestingly, the antibacterial activity of AF was stronger at a highly acidic environment (pH 2) and in a moderate alkalinity range (pH 8–10) compared with a neutral pH (pH 7). These results showed that AF has a good acid-base tolerance.

Effects of enzyme digestion on the antibacterial activity of AF

We then tested the stability of the antibacterial activity of AF in 10 different enzymes commonly found in food and human body environments. The results showed that papain was the only tested enzyme that could eradicate the antibacterial activity of AF, and the other nine applied enzymes had negligible effects on the antibacterial activity of AF (Fig. 5A), indicating that AF has strong enzyme tolerance. To further investigate the sensitivity of AF to papain, we performed a time-based bacteriostatic activity analysis of papain treatment (Fig. 5B). As expected, the antibacterial activity of AF decreased gradually with the increase in time of enzymatic hydrolysis, indicating that the inactivation effect of papain was time-dependent. Based on these results, it is logical to speculate that the active antibacterial metabolite in AF is proteinaceous or peptide in nature.

Spectrum of antibacterial activity of AF

The AF exhibited varying degree of antimicrobial activity when further tested against 20 indicator bacteria and three fungi (Table 3): strong inhibitory activity against Staphylococcus aureus (ATCC 12600); moderate inhibitory activity against Pseudomonas fluorescens CICC 21620 and Pseudomonas aeruginosa ATCC 47085; weak inhibitory against Actinobacillus actinomycetemcomitans BNCC 336945, Listeria monocytogenes ATCC 15313, and Bacillus cereus CICC 10277; and no inhibitory activity against the three tested fungi and some common lactic acid bacteria and/or probiotic bacteria such as L. plantarum P-8, L. rhamnosus Probio-M9, P. acidilactici PA-19, B. animalis subsp. lactis Probio-M8 and L. paracasei Zhang

Table 3

Antibacterial activity spectrum of *Lacticaseibacillus paracasei* Zhang active fraction
Indicator strain	Strain identification number	Inhibition activity
Gram-negative bacterium
Actinobacillus actinomycetemcomitans	BNCC 336945	+
Escherichia coli	CICC 23657	-
Fusobacterium nucleatum subsp. polymorphum	BNCC 336949	-
Porphyromonas gingivalis	BNCC 337441	-
Pseudomonas aeruginosa	ATCC 47085	++
Pseudomonas fluorescens	CICC 21620	++
Riemerella anatipestifer	-	-
Salmonella enterica	CICC 10982	-
Vibrio harveyi	CGMCC 1.8690	-
Vibrio parahemolyticus	CICC 21617	-
Gram-positive bacterium
Bacillus cereus	CICC 10277	+
Bifidobacterium animalis subsp. lactis	Probio-M8	-
Clostridium perfringens	CICC 22949	-
Lacticaseibacillus paracasei	Zhang	-
Lacticaseibacillus rhamnosus	Probio-M9	-
Lactiplantibacillus plantarum	P-8	-
Listeria monocytogenes	ATCC 15313	+
Pediococcus acidilactici	PA-19	-
Staphylococcus aureus	ATCC 12600	+++
Streptococcus mutans	BNCC 336931	-
Fungus
Aspergillus flavus	CICC 2219	-
Candida albicans	CICC 32819	-
Cladosporium sphaerospermum	CICC 2477	-

Remarks: “+++”, “++”, and “+” represent inhibition zone > 15 mm, 11–15 mm, and 7–11 mm, respectively; “-” means no inhibition.

Mining for L. paracasei Zhang bacteriocin gene cluster

BAGEL4 is an online platform that facilitate data mining of bacterial (meta-)genomic DNA for bacteriocins, as well as other bacterial ribosomally synthesized and post-translationally-modified peptides [34]. The BAGEL4 search predicted two areas of interest (AOI), where putative gene clusters for antibacterial substances biosynthesis are located in L. paracasei Zhang genome (Fig. 6). Noteworthy, these potential bacteriocins are possibly involved in the antagonistic activities reported in this work.

AOI_1 (chromosome position from 2079925 to 2100039) contained 52 open reading frames (ORFs) (Table 4). One core gene was predicted to share 50.98% homology with LESI_2163, and a lantibiotic modifying enzyme-encoding gene (LasC) harboring a PF00733 domain was predicted. BLASTP analysis indicated that this gene encodes a protease that catalyzes the conversion of aspartate to asparagine. Other predicted gene features in AOI_1 included transcription regulatory factors, transposase, and phosphotransferase system-related genes.

AOI_2 (from 2311300 to 2335548 contained 72 ORFs with two potential bacteriocins genes as the core genes (Table 4). The two genes were predicted to share 64.00% and 31.82% homology with two bacteriocin-encoding genes, namely enterocin_X_chain_beta and carnocin_CP52, respectively. Besides, within AOI_2, several other ORFs (OR00030, ORF00054, and ORF00056) showed homology to some known class II bacteriocin genes with double-glycine leader peptide; and ORF00030 was homologous with ComC bacteriocin. Other bacteriocin-related gene features were also identified within the AOI_2 region, including: ORF00013, encoding a putative bacteriocin immunity protein, characterized by its specific spatial conformation and functional structural motifs; ORF00007 and ORF00008, encoding a histidine kinase associated with bacteriocin production and a PlnD-liked response regulator for negative regulation of bacteriocin synthesis, respectively; ORF00038, encoding a cognate immunity protein-containing CAAX-like protease, which was homologous to PlnI (P71468_LACPL). Moreover, the region of AOI_2 also encoded a putative ABC transporter gene and a helper factor gene that were homologous to LanT and HlyD, respectively.

Table 4

Functional annotation of gene clusters predicted by BAGEL4
No.	Functional	Accession (Id)
AOI_1
ORF00005	D-lactate dehydrogenase	sp\|Q88VJ2\|LDHD_LACPL
ORF00006	Probable N-acetyl-LL-diaminopimelate aminotransferase	sp\|P16524\|DAPX_BACSU
ORF00013	Uncharacterized protein M6_Spy0233	sp\|Q5XDZ5\|Y233_STRP6
ORF00018	Uncharacterized HIT-like protein MG132	sp\|P47378\|YHIT_MYCGE
LasC	Asparagine synthetase [glutamine-hydrolyzing] 1	sp\|P54420\|ASNB_BACSU
142.2;LESI_2163	—	—
ORF00030	Phosphatidylglycerol lysyltransferase	sp\|C0H3X7\|MPRF_BACSU
ORF00041	1,4-dihydroxy-2-naphthoate octaprenyltransferase	sp\|P39582\|MENA_BACSU
ORF00042	Hexaprenyl-diphosphate synthase large subunit ((2E,6E)-farnesyl-diphosphate specific)	sp\|O66129\|HEXB_MICLU
ORF00045	Acetate kinase	sp\|Q03CP2\|ACKA_LACP3
ORF00049	Uncharacterized transposase-like protein HI_1721	sp\|O05086\|Y1721_HAEIN
ORF00051	Insertion element IS1223 uncharacterized 20.7 kDa protein	sp\|Q48585\|YI3A_LACJH
AOI_2
LanT	ATP-binding protein ComA	sp\|P59653\|COMA_STRR6
ORF00007	Bacteriocin production related histidine kinase	Q1U9H1_LACRE
ORF00008	response regulator PlnD	CAA64207.1
ORF00013	ggmotif; Lactococcin	—
97.2;Enterocin_X_chain_beta	ComC; Lactococcin; Bacteriocin_IIc	—
ORF00030	ComC; Bacteriocin_IIc	—
ORF00038	P71468_LACPL PlnI (Immunity protein PlnI, membrane-bound protease CAAX family)	P71468
97.2;Enterocin_X_chain_beta	Bacteriocin_IIc	—
51.2;Carnocin_CP52	Carnocin	—
ORF00050	Probable maltose O-acetyltransferase	sp\|P37515\|MAA_BACSU
ORF00054	Bacteriocin_IIc	—
ORF00056	Bacteriocin_IIc	—
ORF00059	Oxidoreductase YdhF	sp\|P76187\|YDHF_ECOLI
ORF00061	Uncharacterized oxidoreductase SSP1627	sp\|Q49WS9\|Y1627_STAS1
ORF00063	Probable NADH-dependent flavin oxidoreductase YqiG	sp\|P54524\|YQIG_BACSU
ORF00066	Fumarate hydratase class II	sp\|Q929E8\|FUMC_LISIN
ORF00072	Divalent metal cation transporter MntH	sp\|Q38UX8\|MNTH_LACSS

During the growth and metabolism of lactic acid bacteria, a variety of bioactive metabolites, such as short-chain fatty acids, organic acids, diacetyl, peptides, hydrogen peroxide, extracellular polysaccharides, and bacteriocins, are produced and extracellularly secreted. They play import roles in cellular signaling, and they are the bioactive metabolites that confer probiotic effects on the host [35]. This study preliminarily purified and characterized the antibacterial AF in the CFS of L. paracasei Zhang. We also mined the genome of L. paracasei Zhang for genomic regions encoding putative bacteriocins.

Normally, the concentrations of some active metabolites are too low to be detected by traditional in vitro assays [36, 37]. Therefore, it is necessary to enrich the bioactive fraction for further experiments. Herein, a two-step purification was implemented by ultrafiltration and Sephadex G-25 gel filtration chromatography, which mainly desalted the CFS and concentrated the active metabolites based on size. By testing the antibacterial activity of the ultrafiltrates, we found that the bioactivity was concentration in the 1–3 kDa fraction, which was further purified by gel filtration chromatography and subsequent analysis. In the purification process, small size metabolites like lactic acid and acetic acid would be separated from the active metabolites, as most of these interfering molecules, ions, and acid salts had a molecular size of small than 200 Da. Moreover, we also ensure the osmotic pressure of the diluted AF was in a range not interfering bacterial growth in the agar antimicrobial assays.

Our further assay on the antimicrobial spectrum of AF revealed a broad antibacterial spectrum, inhibiting both members of the Gram-positive (Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus) and Gram-negative (Pseudomonas fluorescens, Pseudomonas aeruginosa, and Actinobacillus actinomycetemcomitans) bacteria. The AF exhibited the strongest inhibitory activity against S. aureus compared with other tested bacteria. S. aureus is both a food and human pathogen. As a foodborne pathogen, S. aureus is widely distributed in air, water, and different kinds of food, and can cause food poisoning by secreting enterotoxin. On the other hand, S. aureus is also widely distributed on skin surface, larynx, nasal cavity and other mucosal surfaces; it does not only cause skin infection, but also induce infective endocarditis, fasciitis, osteomyelitis, and pneumonia occasionally, posing a great threat to human health and safety [37]. Thus, the strong inhibitory activity of AF against S. aureus is of interest for it to be applied in food preservation and products like cosmetics. Another feature of AF is that it exerted no inhibitory effect on lactic acid bacteria or probiotics, which is similar to the antibacterial spectrum of other lactic acid bacteria-originated bacteriocins [38, 39]. This makes AF a desirable food biopreservative, which would not alter the endogenous and beneficial gut microbiota when applied in food.

Apart from a wide antibacterial spectrum, a suitable bacteriocin should ideally have good tolerance to heat, acid-base fluctuation, and common enzymes like proteases [40]. We found that the antibacterial activity of AF has a high stability over a wide range of temperature (40℃ to 100℃) and pH (pH 2–3 and pH 6–10). The good thermal and acid-base stability makes it a suitable biopreservative for harsh and complex industrial production processes. Additionally, the bioactivity of AF was highly resistant to digestion by a variety of common industrial use enzymes and enzymes present in animal/human body (such as trypsin, pepsin, α-amylase and protease K), except for papain, which differs from the high susceptibility of many previously characterized bacteriocins and most antibacterial peptides or bacteriocins towards enzyme inactivation [23, 38, 39, 41, 42]. Trypsin and pepsin are two of the most important digestive proteases existing in the human gastrointestinal tract, and protease K and α-amylase are often used in the food industry. The time-based kinetic analysis of papain digestion of the AF in the current study revealed that papain inactivation was a gradual process, suggesting that the nature of the antibacterial metabolite is protein or peptide. In conclusion, the good stability and strong resistance to different kinds of enzymes make AF suitable for use in food preservation and as an oral supplement against gastrointestinal pathogens [43].

In this study, several putative bacteriocin-like genes were identified in L. paracasei genome with the BAGEL 4 web server, including LESI_2163 in AOI_1, and enterocin_X_chain_beta ( Evalue = 1.88e^− 09, match = 64.000% ), carnocin_CP52 ( Evalue = 1.39e^− 20, match = 31.818% ), ORF13, ORF30, ORF54 and ORF56 in AOI_2. Previous studies have shown that the mature peptide of LSEI_2163 was a class IId bacteriocin that exhibited antimicrobial activity against some lactobacilli and several Listeria species [44]. The sequence of LSEI_2163 was found to be 100% identical to those sequences present in several strains of L. paracasei, including ATCC 334 (accession number, CP000423.1), TD 062 (accession number, CP044361.1), and TCS (accession number, CP038153.1). Enterocin X_chain_beta, a class IIc bacteriocin, belongs to the Lactococcus protein-like family, and has 50.98% homology with Enterocin X from Enterococcus faecium KU-B5. Enterocin X is a heat-resistant dipeptide bacteriocin, contains non-thiopeptides and its full antibacterial activity requires the interaction of two complementary peptides [45]. BLASTN analysis showed that the amino acid sequence was 100% identical to that of L. paracasei (CP032637.1). Carnocin_CP52 is homologous to carnobacteriocin B2, which is the first bacteriocin identified from a strain of C. piscicola isolated from a dairy product and identified as class II bacteriocin [46]. Blast results by UniRef90 revealed that ORF13 belongs to Lactococcin-like family and contains ggmotif, and ORF30, ORF54 and ORF56 are class II bacteriocins containing double-glycine leader peptide. As is known to all, cell density-dependent gene expression in bacteria exists widely and is mediated by extracellular communication molecules. Previous studies have found that Gram-positive bacteria usually perceive population density through post-translational processing of peptide pheromones [47]. The Class II bacteriocins are usually produced as propeptides, which contain a characteristic amino-terminal leader sequence called a double-glycine leader sequence [48]. Three components involve in the regulation of class II bacteriocin production: an inducible peptide, also known as a pheromone, and a two-component regulatory system [49]. Similar to the bacteriocin peptides, the inducing peptide is cationic, synthesized as a propeptide with a double-glycine leader sequence, which is cleaved during transportation. The two-component system includes a membrane-bound histidine protein kinase that plays as an environmental sensor, and a cytoplasmic response regulator that is a DNA-binding protein responsible for the activation of the transcription of its target genes. After secretion, the inducing peptide is subsequently sensed by the dedicated two-component system, thereby inducing the transcription of the operons involved in bacteriocin production [47]. As we know, functional bacterial gene clusters for producing extracellular cationic peptide-bacteriocins are usually organized as operons that comprise a complete sets of genes for production until externalization of the bacteriocin, including at least four gene components: bacteriocin structural gene, specific immune protein gene, ABC transporter gene, and its accessory protein gene [50]. Therefore, based on the predicted gene function, it is likely that AOI_02 but AOI_01 was functional in biosynthesizing bacteriocins that are responsible for the seen antibacterial activity of L. paracasei Zhang subjected to the growth environment and quorum sensing activity. What’s more, amino acid sequence alignment results showed that all of ORF13, ORF30, ORF54, and ORF56 had lower similarity with the reported bacteriocins of other strains of L. paracasei, including L. paracasei ZFM54 [23], L. paracasei LS-6 [51], L. paracasei HD1-7 [52], indicating the most possibility of production of novel bacteriocins by L. paracasei Zhang.

Apart from the conventional use of bacteriocin as food preservative, it has been proposed to expand the applications of bacteriocins from food to health. For example, nisin has shown nisin a cytotoxic effect on SW480 cancer cell line, inducing apoptosis by increasing the ratio of bax/bcl-2 on both mRNA and protein levels [53]. Bacteriocins have also been proposed as potential anti-cancer agents due to their selective action against cancer cells based on distinctive cell membrance differences between healthy and cancer cells. Moreover, antibacterial metabolites, including extracellular polysaccharides, bacteriocins and antimicrobial peptides, have been found to exert beneficially regulate the gut microbiota composition, improve host immune response, and enhancing intestinal barrier function. For example, a study found that adding AMP Gal-13 to the diet of broilers could improve the intestinal digestive capacity, antioxidant activity, and immune function of broilers, ultimately promoting the growth of broilers [54]. These active metabolites can stimulate tissue development and affect the nutritional level and physiological function of the body. Another study found that adding nisin into the diet of broilers could reduce potential jejunal and cecal pathogens, such as Clostridium perfringens and Enterobacteriaceae, and substantially suppress jejunal bacterial fermentation [55]. The addition of nisin and Chinese gallnut to the diet of carp substantially remodulated the intestinal microbiota, suppressing amoeba and enhancing Bacteroides [56].

The L. paracasei Zhang strain has shown multiple probiotic functions in animal and human intervention trials, including the enhancement of antioxidation and anti-lipid peroxidation effects [26], stimulating cellular and humoral immunity and tumor immunity [27], improving blood lipids and liver lipid metabolism [28], preventing type Ⅱ diabetes, protecting liver and preventing liver injury [29], and regulate the gut microbiota via increasing the beneficial microbes, while reducing potential pathogens [26]. In this study, we only confirmed that the CFS of L. casei Zhang contained antibacterial metabolites, probably class II bacteriocins, speculated based on genomics prediction, the molecular size and physico-chemical properties (acid-base, thermos-, and enzyme tolerance) of the bioactivity. Whether these metabolite(s) and/or bacteriocin serve other biological functions in vitro and in vivo remain to be further explored. Future studies should also focus on large-scale purification and identification of the bioactive metabolites, exploring their effectiveness in food preservation and suppression of gastrointestinal infection by pathogens, and elucidating the mechanism of the bioactivity.

From these characteristics, it can be concluded that the active substance in AF is a novel antibacterial metabolite. It is believable that the two outstanding advantages will promote and broaden its application in foods preservation and pathogen infection.

This study preliminarily purified and confirmed that the 1 to 3 kDa fraction of CFS of L. paracasei Zhang exhibited a broad spectrum of antibacterial activity against multiple foodborne and human pathogens, including S. aureus and L. monocytogenes. Based on the genomics prediction and the physico-chemical nature of the antibacterial activity, the bioactive metabolite is possibly a class II bacteriocin. The bioactivity has good tolerance to heat, acid-base change, and digestion by various enzymes, making it a potential candidate for use in food preservation and agent for suppressing gastrointestinal infections.

Author Contributions Tian Huang: Methodology, validation, visualization, formal analysis, writing — original draft, and data curation. Zhaojie Li: Methodology, validation, formal analysis, writing — original draft. Xinan Qu: Validation. Guoqiang Yao: Resources. Lai-Yu Kwok: writing — review and editing. Qiuwen He: Funding acquisition. Heping Zhang: Conceptualization, project administration, and supervision. All authors have read and agreed to the published version of the manuscript.

Funding This work was supported by the National Key R&D Program of China (2022YFD2100700), the National Natural Science Foundation of China (U22A20540), the National Natural Science Foundation of China (32001711), the Inner Mongolia Science and Technology Major Projects (2021ZD0014) and the earmarked fund for CARS36.

Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability The authors confirm that the data supporting this study are available within the article.

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Preliminary purification and partial characterization of a functional bacteriocin of Lacticaseibacillus paracasei Zhang and mining for its gene cluster

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Abstract

Figures

Introduction

Materials and Methods

Microbial strains and cultivation

Antibacterial activity assay

Fractionation of CFS by ultrafiltration

Gel filtration of 1–3 kDa CFS fractions

Characterization of the antibacterial material in AF

Heat stability

pH stability

Enzyme resistance

Results

Preliminary purification of the 1–3 kDa fraction

Characterization of the antibacterial activity in AF

Effect of heat on the antibacterial activity of AF

Effect of pH on the antibacterial activity of AF

Effects of enzyme digestion on the antibacterial activity of AF

Spectrum of antibacterial activity of AF

Discussion

Conclusion

Declarations

References

Additional Declarations

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