Isolation and Identication of Lactobacillus plantarum C010 and Growth Kinetics of its Batch Fermentation

9 Chilled pork is pursuit by people due to its delicious and delicate taste, but it is still susceptible to 10 microbial contamination even under refrigerated conditions. Consequently, to explore microbial 11 preservatives for chilled pork, in this study, the specific spoilage organism Pseudomonas koreensis 12 PS1 from spoiled chilled pork as the indicator was used to isolate the bacteriocin-producing lactic 13 acid bacteria from the soils and fresh cow dungs. Among six bacteriocin-producing bacteria from 14 182 isolates, the strain C010 with higher-yielding, broad-spectrum, subculture stability and protease 15 (pepsin, trypsin and proteinase K) sensitive was selected and identified as Lactobacillus plantarum 16 based on m orphological, biochemical and 16S rDNA gene sequence analysis. Simultaneously, the 17 crude bacteriocin of L. plantarum C010 was stable under high temperature and ultraviolet conditions. The kinetics of bacterial growth and bacteriocin production of L. plantarum C010 were analyzed in 19 batch fermentation. Bacteriocin was produced throughout the logarithmic growth phase and the 20 Leudeking-piret model could characterize the synthesis of bacteriocin well. This present study 21 indicates that bacteriocin-producing L. plantarum C010 has promising potentials to control the 22 specific spoilage organism and can be used as the bio-preservative in food. 23


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Meat and meat products are increasingly popular because of their ample moisture and nutrients,  Nowadays, food preservation can control the growth of microorganisms mainly by physical, 39 chemical and biological methods to extend food shelf life. Unexpectedly, physical preservation has a 40 very short shelf life, while the long-term use of chemical synthetic preservation is potentially harmful 41 to humans (Bharti et al. 2015;Sidooski 2018). Hence, using biological substances to replace physical 42 and chemical synthesis is an important way to maintain the microbial quality and safety of meat and 43 meat products (Costa et al. 2019). This method can reduce the addition of exogenous chemical 44 synthetic preservatives which will lead to the alleviation of the toxic effects on human body. Lactic 45 acid bacteria (LAB) are generally regarded as safe because of their probiotic properties, and the 46 metabolites they produce, such as organic acids, hydrogen peroxide, diacetyl and bacteriocins, etc., 47 have strong antibacterial properties (Karoline et al. 2018;Lv et al. 2018). Among them, the presence 48 of organic acids can not only inhibit the growth of most microorganisms to extend the shelf life of 49 foods, but also enhances the sensory, quality and flavor of fermented foods. However, its excessive 50 use will produce a cumulative chronic damage to the body. As a small molecule polypeptide 51 synthesized by the ribosome, bacteriocin is a natural antibacterial peptide produced by LAB 52 (Todorov et al. 2019). At lower concentrations, it has strong antibacterial activity and is easy to be 53 digested by the human body because of its protein nature; more importantly, based on its colorless 54 and tasteless characteristics, the addition of bacteriocins has little impact on the sensory quality of 55 food (Cotter et al. 2013;Lee et al. 2020). Besides, bacteriocins can withstand higher thermal stresses 56 and have a wider hydrogen (pH) tolerance potential than other protein antimicrobials preservatives. 57 For these reasons, bacteriocins in food preservation and medical industry have currently a wide range 58 of applications (Gautam and Sharma 2009;Woraprayote 2016). 59 Pseudomonas is a dominant specific spoilage organism in chilled pork. The biofilm formed by 60 this bacteria easily adheres to processing equipment such as steel and glass, thus increasing the risk of 61 contamination of chilled pork during its processing and transportation (Sterniša et al. 2019). 62 Presently, Nisin is the only bacteriocin recognized as a food additive in the world and is widely used 63 in the preservation of vegetables, dairy products, meat products and others. Unfortunately, its 64 antibacterial spectrum is narrow, and its application in meat and meat products preservation is not 65 ideal. The reason is that Nisin alone is not sufficient to prevent the growth of Gram-negative spoilage 66 bacteria, such as Pseudomonas fluorescens. However, up to now, even though many 67 bacteriocin-producing LABs have been developed, there are still few bacteriocins that have inhibitory 68 effects on Gram-negative bacteria (Anastasiadou et al. 2008;Chung et al. 1989;Ercolini et al. 2010;69 Woraprayote 2016). At the same time, due to low yield, unsatisfactory purification effect and 70 uncertain toxicological properties, the majority of bacteriocins can only exist in laboratories for 71 freshness preservation, but cannot be applied to industrial production (Leroy and De 2010; Silva 72 Célia C. G. 2018).

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The search for bacteriocin-producing LABs that inhibits Gram-negative and positive bacteria has 74 a good research and application value for reducing the growth of spoilage organisms on the surface of 75 meat products at low temperatures. In this study, Pseudomonas koreensis PS1 isolated by our 76 research team from spoiled chilled pork (Huang et al. 2013), as a gram-negative dominant spoilage 77 bacteria, was used as the indicator for aiming to screen out the bacteriocin-producing LABs from the 78 soil, fresh cow dungs, and then the inhibitory effect and growth profile of bacteriocin production from 79 Lactobacillus plantarum C010 were studied. The isolated LABs were grown in MRS medium and cultured aerobically at 37 ℃. After 24 h of 101 cultivation, cells were centrifuged at 4 ℃, 10 000 rpm for 10 min and divided into two parts.

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One part was adjusted to pH 5.5 with 5 M NaOH and HCl. The same pH treatment of lactic acid, 103 acetic acid and hydrochloric acid was used as the positive controls and the MRS medium had no 104 bacteria as negative control. Besides, 28 g (80 % saturation) ammonium sulfate was added to another 105 cell-free supernatant and the precipitation was dissolved by phosphate buffer solution (pH 6.0) 106 (Saelao 2017). The crude antibacterial substance was collected to against P. koreensis PS1 by using 107 the oxford cup diffusion method to determine the antibacterial activity (Li et al. 2019). The same 108 treatment of MRS medium without LAB was used as the control and each treatment was carried out 109 in triplicates.

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The antibacterial activity of crude bacteriocin was used by oxford cup diffusion method using Gram's staining, cell morphology, catalase reaction, sugar fermentation (glucose, fructose, xylose, 127 sucrose, sorbitol, mannitol), and several others (arginine ammonia produce, hydrogen sulfide 128 produce, pigment produce and gas produce). Then, strains were sent to Sangon (Shanghai, China) and 129 the partial sequence of 16S rRNA obtained was compared with the National Center for 130 Biotechnology Information (NCBI) database to evaluate their homology.

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The stability of antibacterial activity of L. plantarum C010

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Aliquots of 1 mL crude bacteriocin were treated with thermostatic incubated for 30 min at 60, 133 80, 100 ℃ and exposed to high pressure condition for 20 min at 121 ℃. Similarly, crude bacteriocins 134 were placed under UV lamp for 5, 10, 20, 30, 40, 60 min. All treatments were applied to evaluate the 135 antibacterial activity against P. koreensis PS1 by using oxford cup diffusion method, and without any 136 treatment served as the control. Each treatment was carried out in triplicates.

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Growth curve and kinetic analysis of L. plantarum C010

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The suspension of L. plantarum C010 was added into fresh MRS medium at a 2 % volume and 139 cultivate at 37 ℃ for 24 h. Samples were taken out every 2 h and the total number of bacteria was 140 measured by plate count method, while the pH, antibacterial activity and reducing sugar were also 141 determined at the same interval. Then, the relationship among bacterial growth, product synthesis and 142 substrate consumption during the batch fermentation of L. plantarum C010 was explored to provide a 143 theoretical basis for the subsequent large-scale fermentation and condition optimization.

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Determination of the total number of bacteria 145 Plate colony counting method was used to determine the total number of bacteria, and the 146 specific method was as follows: Samples of fermentation broth were taken at different time points 147 and diluted at appropriate multiples, then 0.1 mL was spread on MRS medium and evenly coated, and 148 incubated at 37 ℃ until single colony clearly appeared. Subsequently, the number of colonies for 149 50-300 plate were selected for counting and record. All results were expressed as mean value of the inhibitory diameter ± standard deviation.

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In this study, 182 strains with calcium dissolution circles were screened from soils and fresh cow 164 dungs using MRS agar containing 30 % CaCO3 as the initial screening medium for LABs. All these 165 isolates were shown to be gram positive and catalase negative. After eliminating the differences 166 between the morphology and the samples, 6 strains were found to produce antibacterial substances 167 that removed the acidic interference by pH-adjusted and still had an inhibitory on Gram-negative P. 168 koreensis PS1 (Table 1). Interestingly, further re-screened by ammonium sulfate precipitation and the 169 antibacterial activity of antibacterial substance from these strains all had obvious effect on P. 170 koreensis PS1 than the treatment of removing the acid interference. Hence, the crude extract 171 bacteriocin from the precipitation of ammonium sulfate would be used for subsequent experiments.

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In order to obtain crude bacteriocin with good inhibitory effect, the antibacterial activities of 6 175 strains were determined as shown in Table 2  putida BP-01, etc.). In comparison, the crude bacteriocin of strains C010 and C001 showed more 180 significant antibacterial effect against indicators than the other 4 strains. Interestingly, the crude 181 bacteriocins of six isolates did not produce antibacterial effects against yeasts, suggesting that the 182 crude bacteriocin of LABs had no antibacterial effects against some fungus.

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( Table 2 about here) 184 Strain degradation can occur during the subculture, and it is necessary that determine the number 185 of subcultural to ensure the stability of metabolically active substances of LABs. Accordingly, the 186 isolates were analyzed for the genetic stability during subculture, and 6 strains were conducted 5 187 passages to evaluate the stability of antibacterial activity. Table 3 showed that the bacteriocin 188 produced by 5 strains (R1, N109, C010, J001, C001) maintained great antimicrobial stability after 5 189 generations, especially the strain C010, which had the highest antibacterial activity against P. 190 koreensis PS1 among all 6 strains, and the diameter of inhibition zone more than 14 mm. Whereas 191 strain H011 had poor inhibition stability, and its inhibition effect decreased after three generations.

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The above conclusions collectively suggested that strain C010 had the properties of 193 higher-yielding, broad-antibacterial and stable-subculture, which could be chosen for subsequent 194 testing.

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( Table 3 about here) 196 Sensitivity of antibacterial substance from the strain C010 to enzymes 197 The antibacterial substance degraded by proteases means that they can be digested by human.
198 Table 4 showed that the CFS after the removal of organic acids still had antibacterial effect. A number 199 of studies have reported that LABs can produce a variety of antibacterial substances during 200 metabolism, of which including organic acids, hydrogen peroxide and bacteriocins dominate. Then 201 compared with the unprocessed control, the antibacterial activity of strain C010 treated with catalase 202 did not change significantly, while produced a significant decrease when treated with pepsin, trypsin, 203 proteinase K and the deactivation rate reached 56.59 %, 28.06 %, 77.73 %, respectively. These 204 findings indicated that there was almost no hydrogen peroxide in the antibacterial substances 205 produced by strain C010, but was sensitive to protease. And this feature exhibits the protein property 206 of the antimicrobial substance, and it is speculated that it may be a bacteriocin.

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( Table 4 about  Kong 1995), and the strain C010 could be preliminarily identified as Lactobacillus plantarum (table   214   5). Since phenotypic features are sometimes misidentified, for further confirmation, the partial of 16S 215 rDNA (approximately 1431 bp) sequence was obtained after comparing with the sequences from 216 NCBI 's Gene Bank to confirm the strain C010, and it was found to have 100 % homology with L. 217 plantarum DSM 10667 (Fig.3). Therefore, the strain C010 was identified as L. plantarum C010，and 218 the GenBank access number is MW925129. 219 (Fig. 2, Fig. 3 and table 5 about here) 220 The stability of antibacterial substance from L. plantarum C010

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The stability of crude bacteriocin at different temperatures and UV irradiation were shown in 222   Table 6. The crude bacteriocin of L. plantarum C010 maintained their original activity under high 223 temperature and UV irradiation conditions, which had no significant difference between any 224 treatment and the unprocessed control (P>0.05), and even exposed at 121°C for 20 min. The result 225 demonstrated that crude bacteriocin was performed excellent heat resistance and UV irradiation 226 tolerance, which also laid the theoretical foundation for the subsequent cooperative action of 227 bacteriocin that could be associated with other preservation methods to prolong the freshness of The maximum specific growth rate μm and the initial and maximum total number of bacteria X0 268 and Xm can be obtained by fitting the data nonlinearly with Origin 9.0 through equation (2), and the 269 resulting correlation R 2 was used to assess the degree of fit of the model. And by integrating equation (3), the equation (4) can be obtained and reflects the bacteriocin 282 synthesis versus time:

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A nonlinear fit to the data was performed by Origin 9.0 through equation (4), which allowed the 285 derivation of the constants α, β to determine the model for growth and product synthesis of the 286 bacteriophage, and the resulting correlation R 2 was used to assess the degree of model fit. And by integrating equation (5), the equation (6) can be obtained to reflect substrate 295 concentration versus time:

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A nonlinear fit to the data was performed by Origin 9.0 through equation (6), which obtained the 298 substrate yields Yx and YP of the bacteria and products, and the resulting correlation R 2 was used to 299 assess the degree of model fit.

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Kinetic parameters evaluation 302 The equation (2), (4) and (6) can be evaluated to solve three major models of fermentation 303 kinetics, respectively. The values of each parameter and the correlation coefficient R 2 can be obtained 304 by nonlinear fitting. Besides, the specific parameter values obtained from the above kinetic model are 305 shown in Table 8. After batch fermentation of L. plantarum C010, the growth rate of the strain could 306 be calculated as 0.8239 (h -1 ) by nonlinear fitting, while the predicted initial (X0) and maximum (Xm) 307 total number of bacteria were 2.0736 (10 8 CFU/mL) and 214.55 (10 8 CFU/mL), which were not 308 significantly different from the actual fermentation. Then α and β were obtained as 0.021 8 and 0.000  The models for L. plantarum C010 growth kinetics (7), bacteriocin synthesis kinetics (8)

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In this study, 6 strains, named as J001, N109, R1, H001, C010 and H013, were isolated from 345 soils and cow dungs by using the specific dominant bacteria P. koreensis PS1 as the indicator, and 346 these strains were found to produce antibacterial substances other than organic acids and hydrogen

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(-) represent no inhibit activity.       The colony morphology of the strain C010 on the MRS plate (a), the cell morphology under the optical microscope (1000×) (b) and scanning electron microscope (12000×) (c) Figure 3 The phylogenetic tree of the strain C010 based on 16S rDNA sequence Growth curve and the bacteriocin production of L. plantarum C010 in batch fermentation. Note the differences in the time scale on the x-axis and also the variation in the y-axis scale showing the data. Among them represent pH, represent total bacteria count (×108 CFU/mL), represent reducing sugar content (mg/mL) and the bar chart represent diameter of inhibition zone (mm).

Figure 5
The tting curve model of growth kinetics (a), bacteriocin synthesis (b) and substrate consumption (c) of