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Development of a simple, low-cost and eurytopic medium based on Pleurotus eryngii for lactic acid bacteria

Development of a simple, low-cost and eurytopic medium based on Pleurotus eryngii for lactic acid... Lactic acid bacteria (LAB) are a group of important beneficial microorganisms for human, but their growth is restricted to the habitats with rich nutrients. In order to develop a simple, low-cost and efficient medium based on the mush- room Pleurotus eryngii, this study evaluated the effects of different treatment methods for the mushroom, concentra- tion of the mushroom, buffers, tween 80, MgSO ·7H O, MnSO ·4H O, CuSO ·5H O, riboflavin and ascorbic acid on 4 2 4 2 4 2 the growth of Lactococcus lactis subsp. lactis SLPE1-3. An optimized medium was developed, which was composed of the mushroom at 200 g/L, the buffer sodium acetate at 5 g/L, and riboflavin at 0.5 mg/L. The mushroom was ground, boiled and filtered for the filtrate in advance. In this optimized medium which was named as PSR medium, the population density of SLPE1-3 sharply reached 2.13 × 10 CFU/mL within 18 h of incubation, and still maintained 1.17 × 10 CFU/mL at 120 h. In addition, this study found that 6 kinds of LAB could grow almost well, and maintained high survival in PSR medium compared to M17 or MRS medium, including Lactococcus lactis subsp. lactis, Lactobacil- lus plantarum, Lactococcus lactis subsp. cremoris, Lactobacillus paracasei, Pediococcus pentosaceus and Lactobacillus rhamnosus. These results showed that PSR medium was a simple, low cost and eurytopic medium for the cultivation of LAB, and could replace MRS or M17 medium in the food industry, biomedicine and laboratory. Keywords: Lactic acid bacteria, Pleurotus eryngii, Nutrient requirements, Sodium acetate, Riboflavin postharvest mushroom (Wang et  al. 2008; Daniel et  al. Introduction 2011; Zhao et al. 2013). Lactic acid bacteria (LAB) are a diverse group of Gram- During the last two decades, LAB are extensively positive, nonsporulating, low G + C content bacteria that studied and used in food technology and biomedicine include cocci and bacilli, such as Lactococcus, Lactobacil- because of their commercial potential, especially Lac- lus, Streptococcus, Pediococcus and Leuconostoc (D’Souza tococcus lactis and some species of the Lactobacillus et al. 2012; Wyszyńska et al. 2015). Many bacteria of this genus (Aller et al. 2014; Wyszyńska et al. 2015). But these group are long known as starters of dairy, plant, and meat microorganisms are fastidious in nutrient requirements, fermentations, and could improve the taste and texture and their growth is restricted to the habitats with rich of the fermented foods (Price et  al. 2012). Thus they are nutrients (van Niel and Hahn-Hägerdal 1999; Price et al. usually retained as beneficial microorganisms for human 2012). During the long process of evolution, LAB lost a and animal (Wyszyńska et  al. 2015). But it is worth- number of genes for the biosynthesis of cofactors, and while to mention that some strains of LAB could also lack various biosynthetic pathways for important nutri- cause serious diseases in neonates, aquatic animals and ents, especially amino acids and vitamins (van Niel and Hahn-Hägerdal 1999; Kelly et  al. 2010). Therefore, most of LAB required rich nutrients for their well growth. It is very difficult to develop a generally applicable defined *Correspondence: fqliu20011@sina.com Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, medium for these organisms. Currently, the rich and Nanjing 210014, China undefined media are extensively used for culturing LAB, Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Zhao et al. AMB Expr (2016) 6:65 Page 2 of 8 such as MRS medium (De Man et  al. 1960) and M17 10  g/L, yeast extract 5  g/L, dextrose 20  g/L, polysorb- broth (Terzaghi and Sandine 1975). But these media are ate 80 1 mL/L, ammonium citrate 2 g/L, sodium acetate composed of diversiform materials, and their prices are 5  g/L, magnesium sulfate 0.1  g/L, manganese sulfate very high, such as yeast extract, peptone, polypeptone, 0.05  g/L, dipotassium phosphate 2  g/L, pH 6.8). The soy peptone, beef extract. bacterial culture was used as inoculum in the following Pleurotus eryngii is a kind of favored oyster mushroom, experiments. and contains rich nutrients, such as protein, fiber, car - bohydrates, vitamins and minerals (Cohen et  al. 2002; Eec ff t of different treatment methods for P. eryngii on the Li and Shah 2015). Li and Shah (2015) found that poly- growth of L. lactis subsp. lactis saccharide extracted from P. eryngii could promote the In this study, the fresh mushroom P. eryngii was bought proliferation of Streptococcus thermophilus in fermented from a super vegetable wholesale market in Nanjing, milk, and enhance its viability rate during refrigerated Jiangsu Province, China. The mushroom was cut into storage at 4 °C (Li and Shah 2015). In the previous study, small pieces (1.5–2.0  cm ) for this experiment. This we found that L. lactis subsp. lactis could quickly prolif- experiment included three treatments: (1) 200  g of the erate on the surface of post-harvest P. eryngii, and cause mushroom was ground in a blender (MJ-M176P, Pana- water-soaked lesions (Zhao et al. 2013). sonic Limited, Malaysia) for 3  min at high speed with Currently, P. eryngii has been extensively cultivated 400  mL water. The mushroom slurry was supplemented in the world (Zhao et  al. 2013; Li and Shah 2015). Dur- with 500  mL water, boiled for 20  min, and then filtered ing the cultivation, there is plentiful low-cost inferior through two layers of medical gauze. The final volume of mushroom. The objective of this research was to develop the filtrate was increased to 1000  mL by supplemented a low-cost, simple and eurytopic medium based on the water; (2) 200  g of the mushroom was ground accord- inferior P. eryngii, which could replace MRS or M17 ing to the above description, and then filtered through broth medium for cultivating various LAB. two layers of medical gauze. The final volume of the fil - trate was increased to 1000  mL by supplemented water; Materials and methods (3) 200  g of the mushroom were boiled for 20  min, and Bacterial strains and growth conditions then filtered through two layers of medical gauze. The All bacterial strains used in this study and their origin final volume of the filtrate was increased to 1000  mL by are listed in Table  1. Six strains of them were isolated supplemented water. The pH values were adjusted to from different habitats by our laboratory, including L. 6.8  ±  0.1, respectively. These media were sterilized for lactis subsp. lactis SLPE1-3, Lactobacillus plantarum 20 min at 121 °C. P13, Streptococcus thermophilus M1-6, Lactobacillus Subsequently, 250 μL of L. lactis subsp. Lactis SLPE1-3 paracasei FM-LP-4, Leuconostoc mesenteroides JX5 and culture was added to 50  mL of the above-mentioned fil - Pediococcus pentosaceus SR2-6. L. lactis subsp. cremoris trate media respectively, and incubated for 72 h at 30 °C MG1363 was donated by Prof. Lixin Luo (South China without shaking. The population dynamics of SLPE1-3 University of Technology, China). Lactobacillus rham- was investigated after 0, 3, 9, 18, 30, 48 and 72 h of incu- nosus GG was purchased from American Type Culture bation by the gradient dilution method. In brief, each Collection (ATCC, America). Unless otherwise stated, bacterial culture was diluted with sterile water, and the all bacterial strains were grown at 30 °C for 18 h in MRS diluents were plated on MRS agar plates respectively. The medium (proteose peptone at 10  g/L, beef extract at number of bacterial colonies on each plate was counted Table 1 Lactic acid bacteria (LAB) strains used in this study Strain Origin Source or reference Lactococcus lactis subsp. lactis SLPE1-3 (CGMCC12634) Pleurotus eryngii This laboratory Lactobacillus plantarum P13 Fermented vegetables This laboratory Lactococcus lactis subsp. cremoris MG1363 Plasma cured strain NCDO 712 Jensen and Hammer (1993) Streptococcus thermophilus M1-6 Dairy This laboratory Lactobacillus paracasei FM-LP-4 (CGMCC8600) Dairy This laboratory Pediococcus pentosaceus SR2-6 Sour meat This laboratory Lactobacillus rhamnosus GG (ATCC53103) Human gastrointestinal tract Gorbach (1996) Leuconostoc mesenteroides JX5 Kefir This laboratory Zhao et al. AMB Expr (2016) 6:65 Page 3 of 8 after incubating for 24  h at 30  °C. Every treatment acetate at a final concentration of 5 g/L. The final volume included three repetitions. of the filtrate was increased to 1000 mL by supplemented water. The seven kinds of additives were added respec - Eec ff t of different concentration of P. eryngii on the growth tively to the filtrate at the following final concentrations: of L. lactis subsp. lactis glucose 5  g/L, tween 80 0.5  ml/L, MgSO ·7H O 0.2  g/L, 4 2 According to the description in the above section, 100, MnSO ·4H O 20  mg/L, CuSO ·5H O 3  mg/L, riboflavin 4 2 4 2 150, 200 and 250  g of the mushroom was respectively 0.5  mg/L, ascorbic acid 0.5  mg/L. These additives were ground to make mushroom slurry, boiled for 20  min, selected based on their previously reported influences and then filtered. The final volume of each filtrate was on the growth of L. lactis (De Man et  al. 1960; van Niel increased to 1000  mL by supplemented water. The pH and Hahn-Hägerdal 1999; Aller et  al. 2014). The filtrate values were initially adjusted to 6.8  ±  0.1, respectively. without additive was as the control. The pH values were These media were sterilized for 20 min at 121 °C. Subse - initially adjusted to 6.8  ±  0.1, respectively. These media quently, 250 μL of L. lactis subsp. lactis SLPE1-3 culture were sterilized for 20 min at 121 °C. was added to 50 mL of the filtrate media respectively, and Subsequently, 250 μL of L. lactis subsp. lactis SLPE1-3 incubated for 72  h at 30  °C without shaking. The popu - culture was added to 50  mL of the above-mentioned fil - lation dynamics of SLPE1-3 was investigated after 0, 9, trate media, respectively, and incubated for 120 h at 30 °C 18, 30, 48 and 72 h of incubation by the gradient dilution without shaking. The population dynamics of SLPE1-3 method. Every treatment included three repetitions. was investigated after 0, 9, 18, 30, 48, 72 and 120  h of incubation by the gradient dilution method. Every treat- Eec ff t of different buffers on the growth of L. lactis subsp. ment included three repetitions. lactis According to the description in the above section, Growth dynamics of eight LAB strains in PSR, MRS and M17 1000 g of the mushroom was ground to make mushroom media slurry, boiled for 20  min, and then filtered. The filtrate Based on the above experimental results, the opti- was equally divided into five parts. Three kinds of buff - mized medium was composed of the mushroom filtrate ers, including to ammonium citrate, sodium acetate and at 200  g/L, sodium acetate at 5  g/L, and riboflavin at dipotassium phosphate, were selected and supplemented 0.5 mg/L. This medium was named as PSR medium. The based on MRS medium (De Man et al. 1960). This experi - pH value was adjusted to 6.8  ±  0.1. Then the medium ment included five treatments: (1) the filtrate was supple - was sterilized for 20 min at 121 °C. mented with ammonium citrate at a final concentration Subsequently, 250  μL of each LAB strain culture was of 2  g/L; (2) the filtrate was supplemented with sodium added to 50  mL of PSR, MRS or M17 medium (Hope acetate at a final concentration of 5  g/L; (3) the filtrate Bio-Technogy, Qingdao, China) respectively, and incu- was supplemented with dipotassium phosphate at a final bated for 120 h at 30 °C without shaking. The population concentration of 2 g/L; (4) the filtrate was supplemented dynamics of each LAB strain was investigated after 0, with ammonium citrate at 2 g/L, sodium acetate at 5 g/L, 3, 9, 18, 30, 48, 72 and 120  h of incubation by the gra- and dipotassium phosphate at 2 g/L; (5) the filtrate with - dient dilution method. Every treatment included three out buffer was as the control. The final volume of each repetitions. filtrate was increased to 1000 mL by supplemented water. The pH values were initially adjusted to 6.8  ± 0.1, respec- Statistical analysis tively. These media were sterilized for 20 min at 121 °C. Every treatment was randomly arranged with three rep- Subsequently, 250 μL of L. lactis subsp. lactis SLPE1-3 licates. All data obtained were subjected to analysis of culture was added to 50  mL of the above-mentioned fil - variance (ANOVA) using SPSS 13.0 software (SPP Inc., trate media, respectively, and incubated for 72 h at 30 °C Chicago, USA). The mean values were compared using without shaking. The population dynamics of SLPE1-3 Tukey’s test at P < 0.05. was investigated after 0, 9, 18, 30, 48 and 72  h of incu- bation by the gradient dilution method. Every treatment Results included three repetitions. Eec ff t of different treatment methods for P. eryngii on the growth of L. lactis susp. lactis Eec ff t of different additives on the growth of L. lactis subsp. In three different treatments for P. eryngii, the growth lactis of L. lactis susp. lactis SLPE1-3 showed a similar trend In this experiment, 200  g of the mushroom was ground (Fig.  1). The cell density increased sharply within 9  h of to make mushroom slurry, boiled for 20  min, and then incubation, kept a high level from 9 to 30 h of incubation, filtered. The filtrate was supplemented with sodium and then decreased rapidly after 30 of inoculation. But Zhao et al. AMB Expr (2016) 6:65 Page 4 of 8 Eec ff t of different buffers on the growth of L. lactis subsp. lactis MRS medium, which was extensively used for cultur- ing various LAB species, contained three kinds of buff - ers, including ammonium citrate, sodium acetate and dipotassium phosphate. When these buffers were sup - plemented alone or jointly to the P. eryngii medium, the population density of L. lactis susp. lactis SLPE1-3 was significantly higher than that of the control without any buffer, especially alone sodium acetate at the late stage of incubation (Fig.  3a). At 72  h of incubation, the viable count in the medium supplemented with sodium acetate Fig. 1 Eec ff t of different treatment methods for Pleurotus eryngii (5  g/L) was 9.67  ×  10   CFU/mL, which was 403.9 times mushroom on the growth of Lactococcus lactis subsp. lactis SLPE1-3. Filled diamond the mushroom was ground in a blender for 3 min, that of the control. In addition, alone sodium acetate or boiled for 20 min, and then filtered; filled square the mushroom was the jointly buffers showed higher buffer capacity than ground in a blender for 3 min, and then filtered; filled triangle the ammonium citrate and dipotassium phosphate (Fig.  3b). mushroom was boiled for 20 min, and then filtered. Each symbol is Hence, sodium acetate was used as the buffer in the fol - the average of three replicates lowing experiments. the mushroom P. eryngii medium, which was ground and boiled, could improve survival probability compared to other treatment methods at the late stage of incubation. Hence, the mushroom P. eryngii was ground, boiled and filtered for the subsequent experiments. Eec ff t of different concentration of P. eryngii on the growth of L. lactis subsp. lactis In this experiment, there was a positive relationship between the cell density of L. lactis subsp. lactis SLPE1-3 and the concentration of P. eryngii (<200 g/L), especially at the late stage (Fig.  2). But there was not remarkably difference between 200 and 250 g/L of P. eryngii. Hence, 1000  mL medium contained 200  g of the mushroom P. eryngii in the following experiments. Fig. 3 Eec ff t of different buffers on the growth of L. lactis subsp. lactis SLPE1-3 (a) and the pH values of the culture media (b). Filled diamond the control without buffer; filled square sodium acetate at 5 g/L; filled triangle ammonium citrate at 2 g/L; open square dipotassium phos- Fig. 2 Eec ff t of different concentration of P. eryngii mushroom on the phate at 2 g/L; open triangle sodium acetate at 5 g/L, ammonium growth of L. lactis subsp. lactis SLPE1-3. Each symbol is the average of citrate at 2 g/L and dipotassium phosphate at 2 g/L. Each symbol is three replicates the average of three replicates Zhao et al. AMB Expr (2016) 6:65 Page 5 of 8 Eec ff t of different additives on the growth of L. lactis subsp. value might be disadvantageous to the growth and viabil- lactis ity of LAB. In this experiment, we investigated the effect of seven Based on these results, the optimized medium was additives on the growth of L. lactis subsp. lactis SLPE1- composed of the mushroom P. eryngii at 200 g/L, sodium 3. The results showed that these additives could not pro - acetate at 5  g/L, and riboflavin at 0.5  mg/L, which was mote the growth of SLPE1-3 within 72 h after inoculating named as PSR medium. In addition, the physical prop- compared to the control without additives (Fig. 4a). After erty of PSR medium was relatively stable, and there was 48  h of incubation, the population densities of SLPE1-3 not obvious sedimentation and layer phenomena within sharply decreased in the media supplemented difference 30 days of storage at room temperature (Fig. 5). additives, especially glucose, MnSO and CuSO (Fig. 4a). 4 4 But the additives tween 80, riboflavin and ascorbic acid Growth dynamics of eight LAB strains in PSR, MRS and M17 could remarkably delay the contabescence of SLPE1-3 media compared to the control, especially riboflavin. At 120  h To examine the suitability of PSR medium for various of incubation, the population density of SLPE1-3 in the LAB, a total of 8 LAB strains, belonging to seven spe- medium supplemented riboflavin was 8.88 times that of cies, were tested for their growth in PSR, MRS and the control. In addition, the additive glucose prevented M17 media. Three strains of them (L. lactis subsp. lac - the growth of SLPE1-3 at the earlier stage compared to tis SLPE1-3, L. plantarum P13, L. lactis subsp. cremoris the control, and promoted its contabescence at the later MG1363) grew almost as well and remained highly sur- stage. Among seven additives, only glucose significantly vival rates in PSR and M17 media, but their population promoted the decrease in the pH value of the culture densities were sharply decreased after 48  h of incuba- medium compared to the control (Fig. 4b). The lower pH tion in MRS medium (Fig.  6a–c). Two strains of them (L. paracasei FM-LP-4, Pediococcus pentosaceus SR2-6) grew almost as well in PSR and MRS medium, and bet- ter compared to M17 medium (Fig.  6e, f ). One strain of them (Lactobacillus rhamnosus GG) grew better in PSR Fig. 5 Physical form of the liquid PSR medium which was composed of Pleurotus eryngii mushroom at 200 g/L, sodium acetate at 5 g/L, Fig. 4 Eec ff ts of 7 kinds of additives on the growth of L. lactis subsp. and riboflavin at 0.5 mg/L. The mushroom was ground to make lactis (a) and the pH values of the culture media (b). Each symbol is mushroom slurry, boiled for 20 min, and then filtered for the filtrate the average of three replicates in advance Zhao et al. AMB Expr (2016) 6:65 Page 6 of 8 Fig. 6 Growth dynamics of eight lactic acid bacteria strains in PSR, MRS and M17 media. a Lactococcus lactis subsp. lactis SLPE1-3; b Lactobacillus plantarum P13; c Lactococcus lactis subsp. cremoris MG1363; d Streptococcus thermophilus M1-6; e Lactobacillus paracasei FM-LP-4; f Pediococcus pentosaceus SR2-6; g Lactobacillus rhamnosus GG; h Leuconostoc mesenteroides JX5. Filled diamond PSR medium which was composed of P. eryngii mushroom at 200 g/L, sodium acetate at 5 g/L, and riboflavin at 0.5 mg/L; filled square MRS medium; filled triangle M17 medium. Each symbol is the average of three replicates Zhao et al. AMB Expr (2016) 6:65 Page 7 of 8 medium compared to M17 medium, but poorly com- 1999; Aller et  al. 2014). Aller et  al. (2014) founded that pared to MRS medium (Fig.  6g). In addition, two strains riboflavin (B2) was the only essential B-group vitamin for of them (S. thermophilus M1-6, L. mesenteroides JX5) the growth of L. lactis IL1403 in the chemically defined grew poorly in PSR compared to MRS and M17 media media (CDM) experiment. Riboflavin is indispensa - (Fig.  6d, h). These results showed that PSR medium had ble for cellular metabolism because it is the precursor high suitability for the cultivation of many LAB. of coenzymes flavin mononucleotide (FMN) and flavin adenie dinucleotide (FAD) (LeBlanc et  al. 2011; Aller Discussion et al. 2014). The mushroom P. eryngii contains significant The previous studies had demonstrated that 6-8 kinds of concentrations of vitamins, including C, A, B , B , D and 2 1 amino acids must be supplemented in chemically defined niacin (Manzi et  al. 1999; Stajić et  al. 2009). Hence, the media (CDM) for the cultivation of various LAB (Jensen PSR medium based on the mushroom P. eryngii contains and Hammer 1993; Cocaign-Bousquet et  al. 1995; Aller rich vitamins. In this study, though the additive ribo- et al. 2014). Among these amino acids, glutamic acid and flavin could not further promote the growth of L. lactis asparagine were the most important media components susp. lactis SLPE1-3, it delayed the decay of SLPE1-3 in for the growth of many LAB (Aller et  al. 2014). In addi- the culture medium compared to the control without the tion, LAB could grow better in the medium containing additive riboflavin. 18-19 kinds of amino acids (van Niel and Hahn-Hägerdal Rich media MRS and M17 contain adequate amounts 1999). The mushroom P. eryngii contains rich protein of minerals through the use of yeast extract, such as 2+ 2+ 2+ 2+ 2+ and free amino acids, especially aspartic acid, glutamic Fe , Cu , Mg , Zn , Ca , etc. (van Niel and Hahn- 2+ 2+ acid and arginine (Stajić et al. 2009). This study founded Hägerdal 1999). Some studies founded that Mg , Mn 2+ that 6 kinds of LAB could grow very well in PSR medium and Cu could stimulate the growth of LAB (Olsen and when the mushroom P. eryngii was used as only nitro- Qutub 1970; Hansson and Häggström 1984; Loubiere gen source, and their population densities were more et  al. 1997; Aller et  al. 2014). The mushroom P. eryngii than 1  ×  10   CFU/mL at 30  h of incubation. This study contains rich mineral elements, especially Mg, Cu, Mn, K demonstrated that the mushroom P. eryngii could sup- and Ca (Stajić et al. 2009; Akyüz and Kirbag 2010). In this ply enough amino acids for the growth of most species study, the supplementation of mineral elements (MgSO , of LAB. MnSO and CuSO ) could not simulate the growth of L. 4 4 In bioprocesses, the pH value is an important environ- lactis susp. lactis SLPE1-3. This result showed that the mental factor. The influence of pH on the growth and PSR medium based on the mushroom P. eryngii con- metabolic processes of LAB had been extensively studied tained sufficient Mg, Mn and Cu, and excessive mineral (van Niel and Hahn-Hägerdal 1999; Aller et al. 2014). The elements were not conducive to the growth and viability optimum pH for the growth of LAB was usual between of LAB. 6.0 and 6.5. But, LAB produced much lactic acid during Since LAB have been extensively used in food tech- fermentation, which led to a sharp drop of the pH of the nology, biomedicine and scientific experiments, some culture medium (Wyszyńska et  al. 2015). The growth of researchers committed themselves to developing a simple LAB was inhibited when the pH of the culture medium and low-cost medium for the growth of various LAB (De was under 5.5 (van Niel and Hahn-Hägerdal 1999). The Man et  al. 1960; Terzaghi and Sandine 1975; Rodriguez previous study had demonstrated that some buffers could et al. 2010; Aller et al. 2014). During the fermentation of improve the growth and viability of LAB, such as sodium L. lactis CECT-4434 for producing lactic acid and biosur- acetate, ammonium citrate, dipotassium phosphate (De factant, MRS medium could be replaced with two waste Man et al. 1960; Aller et al. 2014). In this study, we found materials: trimming vine shoots as C source, and distilled that sodium acetate was a more suitable buffer for the wine lees as N, P and micronutrient sources (Rodriguez growth and viability of L. lactis susp. lactis SLPE1-3 than et  al. 2010). In this study, PSR medium was only com- ammonium citrate and dipotassium phosphate. Sodium posed of the low-cost mushroom P. eryngii, sodium ace- acetate showed higher buffering ability for the pH of the tate and riboflavin. The cost of raw materials is only 0.8 culture medium than ammonium citrate and dipotas- dollar or so for 1 L of PSR medium in China, but about sium phosphate. This study demonstrated again the lower 2.5 dollars for 1 L of MRS or M17 medium. PSR medium pH of the culture medium remarkably prevented to the could replace MRS or M17 medium for the cultivation of growth LAB, and promoted the death of LAB (Broadbent L. lactis subsp. lactis, L. plantarum, L. lactis subsp. cre- et al. 2010). moris, L. paracasei, P. pentosaceus, L. rhamnosus, etc. Besides amino acids and pH value, many studies had In conclusion, this study developed a kind of simple, demonstrated that several B-group vitamins were essen- low-cost and eurytopic medium (PSR medium). Com- tial for the growth of LAB (van Niel and Hahn-Hägerdal pared to MRS or M17 medium, many LAB could grow Zhao et al. AMB Expr (2016) 6:65 Page 8 of 8 De Man JC, Rogosa M, Sharpe ME. A medium for the cultivation of lactobacilli. almost as well, and remain highly survival rates in PSR J Appl Microbiol. 1960;23:130–5. doi:10.1111/j.1365-2672.1960.tb00188.x. medium. Hence, PSR medium could replace MRS or D’Souza R, Pandeya DR, Hong S. Review: Lactococcus lacits: an efficient gram M17 medium for the cultivation of many LAB in the food positive cell factory for the production and secretion of recombinant protein. Biomed Res. 2012;23(1):1–7. industry, biomedicine and laboratory. Gorbach SL. The discovery of Lactobacillus GG. Nutr Today. 1996;31:25–45. doi:10.1097/00017285-199611001-00002. Hansson L, Häggström MH. Eec ff ts of growth conditions on the activities of Abbreviations superoxide dismutase and NADH-oxidase/NADH-peroxidase in Strepto- LAB: lactic acid bacteria; CGMCC: China General Microbiological Culture coccus lactis. Curr Microbiol. 1984;10:345–51. doi:10.1007/BF01626563. Collection Center; ATCC: American Type Culture Collection; CDM: chemically Jensen PR, Hammer K. Minimal requirements for exponential growth of Lacto- defined media; FMN: flavin mononucleotide; FAD: flavin adenie dinucleotide; coccus lactis. Appl Environ Microbiol. 1993;59:4363–6. ANOVA: analysis of variance. Kelly WJ, Ward LJH, Leahy SC. Chromosomal diversity in Lactococcus lactis and the origin of dairy starter cultures. Genome Bio Evol. 2010;2:729–44. Authors’ contributions doi:10.1093/gbe/evq056. Planning and designing of study: FL, YZ; Experimentation: YZ, YW, RZ; Result LeBlanc JG, Laiño JE, Juarez del Valle M, Vannini V, van Sinderen D, Analysis: YZ, YW, CS; Manuscript Drafting: YZ, ZS, FL. All authors contributed in Taranto MP, Font de Valdez G, Savoy de Giori G, Sesma F. B-group the final approval. All authors read and approved the final manuscript. vitamin production by lactic acid bacteria-current knowledge and potential applications. J Appl Microbiol. 2011;111:1297–309. Author details doi:10.1111/j.1365-2672.2011.05157.x. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nan- Li S, Shah NP. Eec ff ts of Pleurotus eryngii polysaccharides on bacterial jing 210014, China. Institute of Agro-product Processing, Jiangsu Academy growth, texture properties, proteolytic capacity, and angiotensin-l- of Agricultural Sciences, Nanjing 210014, China. converting enzyme-inhibitory activities of fermented milk. J Dairy Sci. 2015;98(5):2949–61. doi:10.3168/jds.2014-9116. Acknowledgements Loubiere P, Cocaign-Bousquet M, Matos J, Goma G, Lindley ND. Influence The authors are very grateful to Prof. Lixin Luo (South China University of Tech- of end-products inhibition and nutrient limitations on the growth nology, China) for kindly providing Lactococcus lactis subsp. cremoris MG1363. of Lactococcus lactis subsp. lactis. J Appl Microbiol. 1997;82:95–100. doi:10.1111/j.1365-2672.1997.tb03302.x. Competing interests Manzi P, Gambelli L, Marconi S, Vivanti V, Pizzoferrato L. Nutrients in edible The authors declare that they have no competing interests. mushrooms—an inter-species comparative study. Food Chem. 1999;65:477–82. doi:10.1016/S0308-8146(98)00212-X. Ethics approval and consent to participate Olsen HC, Qutub AH. Influence of trace elements on the acid productions by This article does not contain any studies concerned with experimentation on lactic cultures. Cult Dairy Prod. 1970;5:12–7. human or animals. Price CE, Zeyniyev A, Kuipers OP, Kok J. From meadows to milk to mucosa—adaptation of Streptococcus and Lactococcus species to Funding their nutritional environments. FEMS Microbiol Rev. 2012;36:949–71. This study was funded by the National Natural Science Foundation of China doi:10.1111/j.1574-6976.2011.00323.x. (31471637). Rodriguez N, Torrado A, Corrado A, Cortés S, Dominguez JM. Use of waste materials for Lactoccus lactis development. J Sci Food Agric. Received: 22 July 2016 Accepted: 18 August 2016 2010;90(10):1726–34. doi:10.1002/jsfa.4008. Stajić M, Vukojević J, Duletić-Laušević S. Biology of Pleurotus eryngii and role in biotechnological processes: a review. Crit Rev Biotechnol. 2009;29(1):55– 66. doi:10.1080/07388550802688821. Terzaghi BE, Sandine WE. Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol. 1975;29:807–13. References van Niel EWJ, Hahn-Hägerdal B. Nutrient requirements of lactococci in defined Akyüz M, Kirbag S. Element contents of Pleurotus eryngii (DC. ex Fr.) Quel. Var growth media. Appl Microbiol Biotechnol. 1999;52:617–27. doi:10.1007/ eryngii grown on some various agro-wastes. Ekoloji. 2010;9(74):10–4. s002530051569. Aller K, Adamberg K, Timarova V, Seiman A, Feštšenko D, Vilu R. Nutritional Wang PC, Lin YD, Liaw LL, Chern RS, Chen SC. Lactococcus lactis subspecies lac- requirements and media development for Lactococcus lactis IL1403. Appl tis also causes white muscle disease in farmed giant freshwater prawns Microbiol Biotechnol. 2014;98:5871–81. doi:10.1007/s00253-014-5641-7. Macrobracbium rosenbergii. Dis Aquat Organ. 2008;79:9–17. doi:10.3354/ Broadbent JR, Larsen RL, Deibel V, Steele JL. Physiological and transcrip- dao01868. tional response of Lactobacillus casei ATCC334 to acid stress. J Bacteriol. Wyszyńska A, Kobierecka P, Bardowski J, Jagusztyn-Krynicka EK. Lactic acid 2010;192(9):2445–58. doi:10.1128/JB.01618-09. bacteria—20 years exploring their potential as live vectors for mucosal Cocaign-Bousquet M, Garrigues C, Novak L, Lindley ND, Loublere P. vaccination. Appl Microbiol Biotechnol. 2015;99:2967–77. doi:10.1007/ Rational development of a simple synthetic medium for the sustained s00253-015-6498-0. growth of Lactococcus lactis. J Appl Microbiol. 1995;79(1):108–16. Zhao Y, Li P, Hu H, Wang Y, Sun Y, Huang K. Postharvest decay of the cultivated doi:10.1111/j.1365-2672.1995.tb03131.x. mushroom Pleurotus eryngii caused by Lactococcus lactis subsp. lactis. J Cohen R, Persky L, Hadar Y. Biotechnological applications and potential of Plant Pathol. 2013;95(2):247–53. doi:10.4454/JPP.V95I2.019. wood-degrading mushrooms of the genus Pleurotus. Appl Microbiol Biotechnol. 2002;58(5):582–94. doi:10.1007/s00253-002-0930-y. Daniel C, Roussel Y, Kleerebezem M, Pot B. Recombinant lactic acid bacteria as mucosal biotherapeutic agents. Trends Biotechnol. 2011;29(10):499–508. doi:10.1016/j.tibtech.2011.05.002. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png AMB Express Springer Journals

Development of a simple, low-cost and eurytopic medium based on Pleurotus eryngii for lactic acid bacteria

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Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
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Abstract

Lactic acid bacteria (LAB) are a group of important beneficial microorganisms for human, but their growth is restricted to the habitats with rich nutrients. In order to develop a simple, low-cost and efficient medium based on the mush- room Pleurotus eryngii, this study evaluated the effects of different treatment methods for the mushroom, concentra- tion of the mushroom, buffers, tween 80, MgSO ·7H O, MnSO ·4H O, CuSO ·5H O, riboflavin and ascorbic acid on 4 2 4 2 4 2 the growth of Lactococcus lactis subsp. lactis SLPE1-3. An optimized medium was developed, which was composed of the mushroom at 200 g/L, the buffer sodium acetate at 5 g/L, and riboflavin at 0.5 mg/L. The mushroom was ground, boiled and filtered for the filtrate in advance. In this optimized medium which was named as PSR medium, the population density of SLPE1-3 sharply reached 2.13 × 10 CFU/mL within 18 h of incubation, and still maintained 1.17 × 10 CFU/mL at 120 h. In addition, this study found that 6 kinds of LAB could grow almost well, and maintained high survival in PSR medium compared to M17 or MRS medium, including Lactococcus lactis subsp. lactis, Lactobacil- lus plantarum, Lactococcus lactis subsp. cremoris, Lactobacillus paracasei, Pediococcus pentosaceus and Lactobacillus rhamnosus. These results showed that PSR medium was a simple, low cost and eurytopic medium for the cultivation of LAB, and could replace MRS or M17 medium in the food industry, biomedicine and laboratory. Keywords: Lactic acid bacteria, Pleurotus eryngii, Nutrient requirements, Sodium acetate, Riboflavin postharvest mushroom (Wang et  al. 2008; Daniel et  al. Introduction 2011; Zhao et al. 2013). Lactic acid bacteria (LAB) are a diverse group of Gram- During the last two decades, LAB are extensively positive, nonsporulating, low G + C content bacteria that studied and used in food technology and biomedicine include cocci and bacilli, such as Lactococcus, Lactobacil- because of their commercial potential, especially Lac- lus, Streptococcus, Pediococcus and Leuconostoc (D’Souza tococcus lactis and some species of the Lactobacillus et al. 2012; Wyszyńska et al. 2015). Many bacteria of this genus (Aller et al. 2014; Wyszyńska et al. 2015). But these group are long known as starters of dairy, plant, and meat microorganisms are fastidious in nutrient requirements, fermentations, and could improve the taste and texture and their growth is restricted to the habitats with rich of the fermented foods (Price et  al. 2012). Thus they are nutrients (van Niel and Hahn-Hägerdal 1999; Price et al. usually retained as beneficial microorganisms for human 2012). During the long process of evolution, LAB lost a and animal (Wyszyńska et  al. 2015). But it is worth- number of genes for the biosynthesis of cofactors, and while to mention that some strains of LAB could also lack various biosynthetic pathways for important nutri- cause serious diseases in neonates, aquatic animals and ents, especially amino acids and vitamins (van Niel and Hahn-Hägerdal 1999; Kelly et  al. 2010). Therefore, most of LAB required rich nutrients for their well growth. It is very difficult to develop a generally applicable defined *Correspondence: fqliu20011@sina.com Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, medium for these organisms. Currently, the rich and Nanjing 210014, China undefined media are extensively used for culturing LAB, Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Zhao et al. AMB Expr (2016) 6:65 Page 2 of 8 such as MRS medium (De Man et  al. 1960) and M17 10  g/L, yeast extract 5  g/L, dextrose 20  g/L, polysorb- broth (Terzaghi and Sandine 1975). But these media are ate 80 1 mL/L, ammonium citrate 2 g/L, sodium acetate composed of diversiform materials, and their prices are 5  g/L, magnesium sulfate 0.1  g/L, manganese sulfate very high, such as yeast extract, peptone, polypeptone, 0.05  g/L, dipotassium phosphate 2  g/L, pH 6.8). The soy peptone, beef extract. bacterial culture was used as inoculum in the following Pleurotus eryngii is a kind of favored oyster mushroom, experiments. and contains rich nutrients, such as protein, fiber, car - bohydrates, vitamins and minerals (Cohen et  al. 2002; Eec ff t of different treatment methods for P. eryngii on the Li and Shah 2015). Li and Shah (2015) found that poly- growth of L. lactis subsp. lactis saccharide extracted from P. eryngii could promote the In this study, the fresh mushroom P. eryngii was bought proliferation of Streptococcus thermophilus in fermented from a super vegetable wholesale market in Nanjing, milk, and enhance its viability rate during refrigerated Jiangsu Province, China. The mushroom was cut into storage at 4 °C (Li and Shah 2015). In the previous study, small pieces (1.5–2.0  cm ) for this experiment. This we found that L. lactis subsp. lactis could quickly prolif- experiment included three treatments: (1) 200  g of the erate on the surface of post-harvest P. eryngii, and cause mushroom was ground in a blender (MJ-M176P, Pana- water-soaked lesions (Zhao et al. 2013). sonic Limited, Malaysia) for 3  min at high speed with Currently, P. eryngii has been extensively cultivated 400  mL water. The mushroom slurry was supplemented in the world (Zhao et  al. 2013; Li and Shah 2015). Dur- with 500  mL water, boiled for 20  min, and then filtered ing the cultivation, there is plentiful low-cost inferior through two layers of medical gauze. The final volume of mushroom. The objective of this research was to develop the filtrate was increased to 1000  mL by supplemented a low-cost, simple and eurytopic medium based on the water; (2) 200  g of the mushroom was ground accord- inferior P. eryngii, which could replace MRS or M17 ing to the above description, and then filtered through broth medium for cultivating various LAB. two layers of medical gauze. The final volume of the fil - trate was increased to 1000  mL by supplemented water; Materials and methods (3) 200  g of the mushroom were boiled for 20  min, and Bacterial strains and growth conditions then filtered through two layers of medical gauze. The All bacterial strains used in this study and their origin final volume of the filtrate was increased to 1000  mL by are listed in Table  1. Six strains of them were isolated supplemented water. The pH values were adjusted to from different habitats by our laboratory, including L. 6.8  ±  0.1, respectively. These media were sterilized for lactis subsp. lactis SLPE1-3, Lactobacillus plantarum 20 min at 121 °C. P13, Streptococcus thermophilus M1-6, Lactobacillus Subsequently, 250 μL of L. lactis subsp. Lactis SLPE1-3 paracasei FM-LP-4, Leuconostoc mesenteroides JX5 and culture was added to 50  mL of the above-mentioned fil - Pediococcus pentosaceus SR2-6. L. lactis subsp. cremoris trate media respectively, and incubated for 72 h at 30 °C MG1363 was donated by Prof. Lixin Luo (South China without shaking. The population dynamics of SLPE1-3 University of Technology, China). Lactobacillus rham- was investigated after 0, 3, 9, 18, 30, 48 and 72 h of incu- nosus GG was purchased from American Type Culture bation by the gradient dilution method. In brief, each Collection (ATCC, America). Unless otherwise stated, bacterial culture was diluted with sterile water, and the all bacterial strains were grown at 30 °C for 18 h in MRS diluents were plated on MRS agar plates respectively. The medium (proteose peptone at 10  g/L, beef extract at number of bacterial colonies on each plate was counted Table 1 Lactic acid bacteria (LAB) strains used in this study Strain Origin Source or reference Lactococcus lactis subsp. lactis SLPE1-3 (CGMCC12634) Pleurotus eryngii This laboratory Lactobacillus plantarum P13 Fermented vegetables This laboratory Lactococcus lactis subsp. cremoris MG1363 Plasma cured strain NCDO 712 Jensen and Hammer (1993) Streptococcus thermophilus M1-6 Dairy This laboratory Lactobacillus paracasei FM-LP-4 (CGMCC8600) Dairy This laboratory Pediococcus pentosaceus SR2-6 Sour meat This laboratory Lactobacillus rhamnosus GG (ATCC53103) Human gastrointestinal tract Gorbach (1996) Leuconostoc mesenteroides JX5 Kefir This laboratory Zhao et al. AMB Expr (2016) 6:65 Page 3 of 8 after incubating for 24  h at 30  °C. Every treatment acetate at a final concentration of 5 g/L. The final volume included three repetitions. of the filtrate was increased to 1000 mL by supplemented water. The seven kinds of additives were added respec - Eec ff t of different concentration of P. eryngii on the growth tively to the filtrate at the following final concentrations: of L. lactis subsp. lactis glucose 5  g/L, tween 80 0.5  ml/L, MgSO ·7H O 0.2  g/L, 4 2 According to the description in the above section, 100, MnSO ·4H O 20  mg/L, CuSO ·5H O 3  mg/L, riboflavin 4 2 4 2 150, 200 and 250  g of the mushroom was respectively 0.5  mg/L, ascorbic acid 0.5  mg/L. These additives were ground to make mushroom slurry, boiled for 20  min, selected based on their previously reported influences and then filtered. The final volume of each filtrate was on the growth of L. lactis (De Man et  al. 1960; van Niel increased to 1000  mL by supplemented water. The pH and Hahn-Hägerdal 1999; Aller et  al. 2014). The filtrate values were initially adjusted to 6.8  ±  0.1, respectively. without additive was as the control. The pH values were These media were sterilized for 20 min at 121 °C. Subse - initially adjusted to 6.8  ±  0.1, respectively. These media quently, 250 μL of L. lactis subsp. lactis SLPE1-3 culture were sterilized for 20 min at 121 °C. was added to 50 mL of the filtrate media respectively, and Subsequently, 250 μL of L. lactis subsp. lactis SLPE1-3 incubated for 72  h at 30  °C without shaking. The popu - culture was added to 50  mL of the above-mentioned fil - lation dynamics of SLPE1-3 was investigated after 0, 9, trate media, respectively, and incubated for 120 h at 30 °C 18, 30, 48 and 72 h of incubation by the gradient dilution without shaking. The population dynamics of SLPE1-3 method. Every treatment included three repetitions. was investigated after 0, 9, 18, 30, 48, 72 and 120  h of incubation by the gradient dilution method. Every treat- Eec ff t of different buffers on the growth of L. lactis subsp. ment included three repetitions. lactis According to the description in the above section, Growth dynamics of eight LAB strains in PSR, MRS and M17 1000 g of the mushroom was ground to make mushroom media slurry, boiled for 20  min, and then filtered. The filtrate Based on the above experimental results, the opti- was equally divided into five parts. Three kinds of buff - mized medium was composed of the mushroom filtrate ers, including to ammonium citrate, sodium acetate and at 200  g/L, sodium acetate at 5  g/L, and riboflavin at dipotassium phosphate, were selected and supplemented 0.5 mg/L. This medium was named as PSR medium. The based on MRS medium (De Man et al. 1960). This experi - pH value was adjusted to 6.8  ±  0.1. Then the medium ment included five treatments: (1) the filtrate was supple - was sterilized for 20 min at 121 °C. mented with ammonium citrate at a final concentration Subsequently, 250  μL of each LAB strain culture was of 2  g/L; (2) the filtrate was supplemented with sodium added to 50  mL of PSR, MRS or M17 medium (Hope acetate at a final concentration of 5  g/L; (3) the filtrate Bio-Technogy, Qingdao, China) respectively, and incu- was supplemented with dipotassium phosphate at a final bated for 120 h at 30 °C without shaking. The population concentration of 2 g/L; (4) the filtrate was supplemented dynamics of each LAB strain was investigated after 0, with ammonium citrate at 2 g/L, sodium acetate at 5 g/L, 3, 9, 18, 30, 48, 72 and 120  h of incubation by the gra- and dipotassium phosphate at 2 g/L; (5) the filtrate with - dient dilution method. Every treatment included three out buffer was as the control. The final volume of each repetitions. filtrate was increased to 1000 mL by supplemented water. The pH values were initially adjusted to 6.8  ± 0.1, respec- Statistical analysis tively. These media were sterilized for 20 min at 121 °C. Every treatment was randomly arranged with three rep- Subsequently, 250 μL of L. lactis subsp. lactis SLPE1-3 licates. All data obtained were subjected to analysis of culture was added to 50  mL of the above-mentioned fil - variance (ANOVA) using SPSS 13.0 software (SPP Inc., trate media, respectively, and incubated for 72 h at 30 °C Chicago, USA). The mean values were compared using without shaking. The population dynamics of SLPE1-3 Tukey’s test at P < 0.05. was investigated after 0, 9, 18, 30, 48 and 72  h of incu- bation by the gradient dilution method. Every treatment Results included three repetitions. Eec ff t of different treatment methods for P. eryngii on the growth of L. lactis susp. lactis Eec ff t of different additives on the growth of L. lactis subsp. In three different treatments for P. eryngii, the growth lactis of L. lactis susp. lactis SLPE1-3 showed a similar trend In this experiment, 200  g of the mushroom was ground (Fig.  1). The cell density increased sharply within 9  h of to make mushroom slurry, boiled for 20  min, and then incubation, kept a high level from 9 to 30 h of incubation, filtered. The filtrate was supplemented with sodium and then decreased rapidly after 30 of inoculation. But Zhao et al. AMB Expr (2016) 6:65 Page 4 of 8 Eec ff t of different buffers on the growth of L. lactis subsp. lactis MRS medium, which was extensively used for cultur- ing various LAB species, contained three kinds of buff - ers, including ammonium citrate, sodium acetate and dipotassium phosphate. When these buffers were sup - plemented alone or jointly to the P. eryngii medium, the population density of L. lactis susp. lactis SLPE1-3 was significantly higher than that of the control without any buffer, especially alone sodium acetate at the late stage of incubation (Fig.  3a). At 72  h of incubation, the viable count in the medium supplemented with sodium acetate Fig. 1 Eec ff t of different treatment methods for Pleurotus eryngii (5  g/L) was 9.67  ×  10   CFU/mL, which was 403.9 times mushroom on the growth of Lactococcus lactis subsp. lactis SLPE1-3. Filled diamond the mushroom was ground in a blender for 3 min, that of the control. In addition, alone sodium acetate or boiled for 20 min, and then filtered; filled square the mushroom was the jointly buffers showed higher buffer capacity than ground in a blender for 3 min, and then filtered; filled triangle the ammonium citrate and dipotassium phosphate (Fig.  3b). mushroom was boiled for 20 min, and then filtered. Each symbol is Hence, sodium acetate was used as the buffer in the fol - the average of three replicates lowing experiments. the mushroom P. eryngii medium, which was ground and boiled, could improve survival probability compared to other treatment methods at the late stage of incubation. Hence, the mushroom P. eryngii was ground, boiled and filtered for the subsequent experiments. Eec ff t of different concentration of P. eryngii on the growth of L. lactis subsp. lactis In this experiment, there was a positive relationship between the cell density of L. lactis subsp. lactis SLPE1-3 and the concentration of P. eryngii (<200 g/L), especially at the late stage (Fig.  2). But there was not remarkably difference between 200 and 250 g/L of P. eryngii. Hence, 1000  mL medium contained 200  g of the mushroom P. eryngii in the following experiments. Fig. 3 Eec ff t of different buffers on the growth of L. lactis subsp. lactis SLPE1-3 (a) and the pH values of the culture media (b). Filled diamond the control without buffer; filled square sodium acetate at 5 g/L; filled triangle ammonium citrate at 2 g/L; open square dipotassium phos- Fig. 2 Eec ff t of different concentration of P. eryngii mushroom on the phate at 2 g/L; open triangle sodium acetate at 5 g/L, ammonium growth of L. lactis subsp. lactis SLPE1-3. Each symbol is the average of citrate at 2 g/L and dipotassium phosphate at 2 g/L. Each symbol is three replicates the average of three replicates Zhao et al. AMB Expr (2016) 6:65 Page 5 of 8 Eec ff t of different additives on the growth of L. lactis subsp. value might be disadvantageous to the growth and viabil- lactis ity of LAB. In this experiment, we investigated the effect of seven Based on these results, the optimized medium was additives on the growth of L. lactis subsp. lactis SLPE1- composed of the mushroom P. eryngii at 200 g/L, sodium 3. The results showed that these additives could not pro - acetate at 5  g/L, and riboflavin at 0.5  mg/L, which was mote the growth of SLPE1-3 within 72 h after inoculating named as PSR medium. In addition, the physical prop- compared to the control without additives (Fig. 4a). After erty of PSR medium was relatively stable, and there was 48  h of incubation, the population densities of SLPE1-3 not obvious sedimentation and layer phenomena within sharply decreased in the media supplemented difference 30 days of storage at room temperature (Fig. 5). additives, especially glucose, MnSO and CuSO (Fig. 4a). 4 4 But the additives tween 80, riboflavin and ascorbic acid Growth dynamics of eight LAB strains in PSR, MRS and M17 could remarkably delay the contabescence of SLPE1-3 media compared to the control, especially riboflavin. At 120  h To examine the suitability of PSR medium for various of incubation, the population density of SLPE1-3 in the LAB, a total of 8 LAB strains, belonging to seven spe- medium supplemented riboflavin was 8.88 times that of cies, were tested for their growth in PSR, MRS and the control. In addition, the additive glucose prevented M17 media. Three strains of them (L. lactis subsp. lac - the growth of SLPE1-3 at the earlier stage compared to tis SLPE1-3, L. plantarum P13, L. lactis subsp. cremoris the control, and promoted its contabescence at the later MG1363) grew almost as well and remained highly sur- stage. Among seven additives, only glucose significantly vival rates in PSR and M17 media, but their population promoted the decrease in the pH value of the culture densities were sharply decreased after 48  h of incuba- medium compared to the control (Fig. 4b). The lower pH tion in MRS medium (Fig.  6a–c). Two strains of them (L. paracasei FM-LP-4, Pediococcus pentosaceus SR2-6) grew almost as well in PSR and MRS medium, and bet- ter compared to M17 medium (Fig.  6e, f ). One strain of them (Lactobacillus rhamnosus GG) grew better in PSR Fig. 5 Physical form of the liquid PSR medium which was composed of Pleurotus eryngii mushroom at 200 g/L, sodium acetate at 5 g/L, Fig. 4 Eec ff ts of 7 kinds of additives on the growth of L. lactis subsp. and riboflavin at 0.5 mg/L. The mushroom was ground to make lactis (a) and the pH values of the culture media (b). Each symbol is mushroom slurry, boiled for 20 min, and then filtered for the filtrate the average of three replicates in advance Zhao et al. AMB Expr (2016) 6:65 Page 6 of 8 Fig. 6 Growth dynamics of eight lactic acid bacteria strains in PSR, MRS and M17 media. a Lactococcus lactis subsp. lactis SLPE1-3; b Lactobacillus plantarum P13; c Lactococcus lactis subsp. cremoris MG1363; d Streptococcus thermophilus M1-6; e Lactobacillus paracasei FM-LP-4; f Pediococcus pentosaceus SR2-6; g Lactobacillus rhamnosus GG; h Leuconostoc mesenteroides JX5. Filled diamond PSR medium which was composed of P. eryngii mushroom at 200 g/L, sodium acetate at 5 g/L, and riboflavin at 0.5 mg/L; filled square MRS medium; filled triangle M17 medium. Each symbol is the average of three replicates Zhao et al. AMB Expr (2016) 6:65 Page 7 of 8 medium compared to M17 medium, but poorly com- 1999; Aller et  al. 2014). Aller et  al. (2014) founded that pared to MRS medium (Fig.  6g). In addition, two strains riboflavin (B2) was the only essential B-group vitamin for of them (S. thermophilus M1-6, L. mesenteroides JX5) the growth of L. lactis IL1403 in the chemically defined grew poorly in PSR compared to MRS and M17 media media (CDM) experiment. Riboflavin is indispensa - (Fig.  6d, h). These results showed that PSR medium had ble for cellular metabolism because it is the precursor high suitability for the cultivation of many LAB. of coenzymes flavin mononucleotide (FMN) and flavin adenie dinucleotide (FAD) (LeBlanc et  al. 2011; Aller Discussion et al. 2014). The mushroom P. eryngii contains significant The previous studies had demonstrated that 6-8 kinds of concentrations of vitamins, including C, A, B , B , D and 2 1 amino acids must be supplemented in chemically defined niacin (Manzi et  al. 1999; Stajić et  al. 2009). Hence, the media (CDM) for the cultivation of various LAB (Jensen PSR medium based on the mushroom P. eryngii contains and Hammer 1993; Cocaign-Bousquet et  al. 1995; Aller rich vitamins. In this study, though the additive ribo- et al. 2014). Among these amino acids, glutamic acid and flavin could not further promote the growth of L. lactis asparagine were the most important media components susp. lactis SLPE1-3, it delayed the decay of SLPE1-3 in for the growth of many LAB (Aller et  al. 2014). In addi- the culture medium compared to the control without the tion, LAB could grow better in the medium containing additive riboflavin. 18-19 kinds of amino acids (van Niel and Hahn-Hägerdal Rich media MRS and M17 contain adequate amounts 1999). The mushroom P. eryngii contains rich protein of minerals through the use of yeast extract, such as 2+ 2+ 2+ 2+ 2+ and free amino acids, especially aspartic acid, glutamic Fe , Cu , Mg , Zn , Ca , etc. (van Niel and Hahn- 2+ 2+ acid and arginine (Stajić et al. 2009). This study founded Hägerdal 1999). Some studies founded that Mg , Mn 2+ that 6 kinds of LAB could grow very well in PSR medium and Cu could stimulate the growth of LAB (Olsen and when the mushroom P. eryngii was used as only nitro- Qutub 1970; Hansson and Häggström 1984; Loubiere gen source, and their population densities were more et  al. 1997; Aller et  al. 2014). The mushroom P. eryngii than 1  ×  10   CFU/mL at 30  h of incubation. This study contains rich mineral elements, especially Mg, Cu, Mn, K demonstrated that the mushroom P. eryngii could sup- and Ca (Stajić et al. 2009; Akyüz and Kirbag 2010). In this ply enough amino acids for the growth of most species study, the supplementation of mineral elements (MgSO , of LAB. MnSO and CuSO ) could not simulate the growth of L. 4 4 In bioprocesses, the pH value is an important environ- lactis susp. lactis SLPE1-3. This result showed that the mental factor. The influence of pH on the growth and PSR medium based on the mushroom P. eryngii con- metabolic processes of LAB had been extensively studied tained sufficient Mg, Mn and Cu, and excessive mineral (van Niel and Hahn-Hägerdal 1999; Aller et al. 2014). The elements were not conducive to the growth and viability optimum pH for the growth of LAB was usual between of LAB. 6.0 and 6.5. But, LAB produced much lactic acid during Since LAB have been extensively used in food tech- fermentation, which led to a sharp drop of the pH of the nology, biomedicine and scientific experiments, some culture medium (Wyszyńska et  al. 2015). The growth of researchers committed themselves to developing a simple LAB was inhibited when the pH of the culture medium and low-cost medium for the growth of various LAB (De was under 5.5 (van Niel and Hahn-Hägerdal 1999). The Man et  al. 1960; Terzaghi and Sandine 1975; Rodriguez previous study had demonstrated that some buffers could et al. 2010; Aller et al. 2014). During the fermentation of improve the growth and viability of LAB, such as sodium L. lactis CECT-4434 for producing lactic acid and biosur- acetate, ammonium citrate, dipotassium phosphate (De factant, MRS medium could be replaced with two waste Man et al. 1960; Aller et al. 2014). In this study, we found materials: trimming vine shoots as C source, and distilled that sodium acetate was a more suitable buffer for the wine lees as N, P and micronutrient sources (Rodriguez growth and viability of L. lactis susp. lactis SLPE1-3 than et  al. 2010). In this study, PSR medium was only com- ammonium citrate and dipotassium phosphate. Sodium posed of the low-cost mushroom P. eryngii, sodium ace- acetate showed higher buffering ability for the pH of the tate and riboflavin. The cost of raw materials is only 0.8 culture medium than ammonium citrate and dipotas- dollar or so for 1 L of PSR medium in China, but about sium phosphate. This study demonstrated again the lower 2.5 dollars for 1 L of MRS or M17 medium. PSR medium pH of the culture medium remarkably prevented to the could replace MRS or M17 medium for the cultivation of growth LAB, and promoted the death of LAB (Broadbent L. lactis subsp. lactis, L. plantarum, L. lactis subsp. cre- et al. 2010). moris, L. paracasei, P. pentosaceus, L. rhamnosus, etc. Besides amino acids and pH value, many studies had In conclusion, this study developed a kind of simple, demonstrated that several B-group vitamins were essen- low-cost and eurytopic medium (PSR medium). Com- tial for the growth of LAB (van Niel and Hahn-Hägerdal pared to MRS or M17 medium, many LAB could grow Zhao et al. AMB Expr (2016) 6:65 Page 8 of 8 De Man JC, Rogosa M, Sharpe ME. A medium for the cultivation of lactobacilli. almost as well, and remain highly survival rates in PSR J Appl Microbiol. 1960;23:130–5. doi:10.1111/j.1365-2672.1960.tb00188.x. medium. Hence, PSR medium could replace MRS or D’Souza R, Pandeya DR, Hong S. Review: Lactococcus lacits: an efficient gram M17 medium for the cultivation of many LAB in the food positive cell factory for the production and secretion of recombinant protein. Biomed Res. 2012;23(1):1–7. industry, biomedicine and laboratory. Gorbach SL. The discovery of Lactobacillus GG. Nutr Today. 1996;31:25–45. doi:10.1097/00017285-199611001-00002. Hansson L, Häggström MH. Eec ff ts of growth conditions on the activities of Abbreviations superoxide dismutase and NADH-oxidase/NADH-peroxidase in Strepto- LAB: lactic acid bacteria; CGMCC: China General Microbiological Culture coccus lactis. Curr Microbiol. 1984;10:345–51. doi:10.1007/BF01626563. Collection Center; ATCC: American Type Culture Collection; CDM: chemically Jensen PR, Hammer K. Minimal requirements for exponential growth of Lacto- defined media; FMN: flavin mononucleotide; FAD: flavin adenie dinucleotide; coccus lactis. Appl Environ Microbiol. 1993;59:4363–6. ANOVA: analysis of variance. Kelly WJ, Ward LJH, Leahy SC. Chromosomal diversity in Lactococcus lactis and the origin of dairy starter cultures. Genome Bio Evol. 2010;2:729–44. Authors’ contributions doi:10.1093/gbe/evq056. Planning and designing of study: FL, YZ; Experimentation: YZ, YW, RZ; Result LeBlanc JG, Laiño JE, Juarez del Valle M, Vannini V, van Sinderen D, Analysis: YZ, YW, CS; Manuscript Drafting: YZ, ZS, FL. 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