A total of 60 lactic acid bacteria (LAB) strains isolated from Chinese traditional food products were screened for antifungal activity. 7 strains showed inhibition activity towards both Mucor sp. and Penicillium sp. isolated from spoiled cheese in the Potato Dextrose Agar well diffusion assay. The production of phenyllactic acid (PLA) was detected. The highest production of PLA was 16.24mg/L, and there's no strict relationship between PLA production and inhibition zone. The lactic acid and acetic acid adjusted to the same pH values in MRS broth as the culture supernatants showed no inhibition activities at all. The antifungal activity of Lactobacillus casei AST18 was investigated. The optimum production of antifungal compound was at 37°C for 48 h and at the initial pH of 6-7. The antifungal compound was sensitive towards pH and heat treatment but it was not affected after treatment of trypsin and pepsin. This research indicted that the antifungal activity is involved with several pH dependent compounds that react simultaneously to inhibit the growth of filamentous fungus.
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Keywords: Antifungal, lactic acid bacteria, Lactobacillus casei, phenyllactic acid, Penicillium sp.
Moulds and yeast are able to grow on almost all kinds of food: cereals, milk, fruits, vegetables, nuts, fats and products of these. O.Filtenborg(1996) concluded that a very limited number of fungal species(2-12) have been associated with the spoilage of each food and they are mainly Penicillium, Aspergillus and Fusarium. Such as: The most important spoilage species of cheese without preservatives added are: Penicillium commune and P. nalgiovense. Now the Biopreservation is wildly used to extend shelf-life and enhanced safety of food obtained by the natural or added microflora or their antimicrobial products(Schnürer, 2005).
Lactic acid bacteria have a long history of use in food products and feeds. LAB can decrease the pH value and produce antibacterial compounds. In the past few years, lots of researches have found that LAB has the ability to inhibit the growth of molds and yeasts. L.A.M. Ryan (2008) used antifungal Lactobacillus plantarum strains to reduce the amount of calcium propionate in bread. A strong synergistic effect occurred when calcium propionate and antifungal sourdoughs were combined into the bread formulation.
Several antifungal compounds have been isolated from bacterial cultures. Magnusson and Schnürer (2001) firstly reported the lactobacillus coryniformis subsp.coryniformis strain produced a broad-spectrum antifungal compound of proteincaceous. Gerez(2009), Magnusson(2003), Ströme(2002,2005), Lavermicocca(2000) have reported the potential antifungal compound as phenyl lactic acid. Rouse (2008) have separated possibly cyclic dipeptide from Pc.pentosaceous. Yang (2010) firstly reported antifungal activity of LAB cyclo(Leu-Leu), a class of 2,5-diketopiperazines. 2,5-diketopiperazines recently have attracted attention due to their biological properties (McCleland et al., 2004). But as antifungal activities in other fractions have been observed they believed that more antifungal compounds were present in addition to the one had been detected.
The aims of the present study were to detect the antifungal activities of LAB which isolated from Chinese traditional food products (pickle and Beijing yoghourt). One of the best strains AFS18 was selected for further study. The culture condition of strain AFS18 and the properties of the metabolite were studied for a further application of the antifungal activities in the preservation of dairy product and other foods. The indictor mold strain was isolated f rom spoilage cheese and identified as penicillium sp.
2. Materials and methods
2.1 Cultures and Media
42 strains were isolated from Chinese traditional dairy products. 18 strains were isolated from pickle. Strains were cultured on deMan Rogosa and Sharpe-MRS agar or in MRS broth (Land Bridge Technology Co., China) at 37â„ƒ for 24h and were maintained for longer storage at -80â„ƒ in MRS supplemented with 25 % (v/v) glycerol.
Fungal isolated and yeast strains
Penicillium sp. and mucor sp. were isolated from spoilage cheese. Strains were maintained on Potato Dextrose Agar (Land Bridge Technology Co., China) at 30â„ƒ for 4 days and stored at 4â„ƒ. Saccharomyces cerevisiaes YS-1, YS-2, YS-3, YS-4, YS-5, YS-6, YS-7 were preserved in our laboratory.
2.2 Preparation of the spore solution
The molds grew on PDA slants at 30â„ƒ for 7days until the sporulation formed. Then the spores were collected after vigorously shaking the slants with sterile water which contain 0.05 %( v\v) Tween-80. The concentration of molds was measured by hemocytometer (Qiujing Company, Shanghai, China) and adjusted to 106 per ml.
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2.3 Preparation of culture supernatants
Lb. casei AST18 was inculcated in MRS broth at 37â„ƒ for 48h. The culture was centrifuged at 4000g for 10min. The supernatants were filtered through a 0.22µm sterile filter (Mili Company, Shanghai, China). The cell free supernatants were used for further experiments.
2.4 Antifungal activity assays
The modified agar well diffusion method (Magnusson and Schnürer, 2001) was used to detect antifungal activity. The penicillium sp. was used as directed molds. For the agar well diffusion method 15ml PDA were added to the plate. Then 100µl spore solution was sprayed on the coagulative PDA. The 7.5mm (outside diameter) wells were settled down for 5min. Then 200µl culture supernatants were added into the wells. The places were incubated at 30â„ƒ for 48h and examined for clear zones of inhibition around the wells with vernier caliper (Shanghai Shengliang Suring tools Co., China ). The sterile MRS broth adjusted to the lowest pH of the culture supernatant was used as a control.
The above experiment was down in duplicate.
2.5 Analysis of organic acids
The concentration of phenyllactic acid (PLA) in the culture was analyzed by High Performance Liquid Chromatography (HPLC) (Aglient Technologies 1200 series). D, L-3-phenyllactic acid was purchased from Sigma-Aldrich. The culture supernatants were prepared as 2.3 mentioned before. PLA was determined using HPLC equipped with an Agilent Zorbax Eclipse-C18 column (4.5mm*150mm, 5µm). Linear gradient elution was used with methanol/0.05% trifluoroacetic acid-solvent A (Sigma-Aldrich) and water/0.05% trifluoroacetic acid-solvent B at 1 mL min-1 and A/B ratios of 10: 90, 100: 0, 100: 0, and 10: 90, with run times of 0, 20, 23, and 25 min, respectively. PLA was detected at 210 nm (Li et al., 2007; Emanuele et al., 2006).
The antifungal activities of lactic acid and acetic acid were determined. The pH values of the 7 LAB cultures supernatants were detected. And the MRS broths with lactic acid or acetic acid were adjusted to the same pH value as the cultures supernatants' respectively. The agar well diffusion assay was used to determine the antifungal activity of the solution.
2.6 Influence of temperature, pH and aeration on production of antifungal acticity
The effect of temperature on antifungal compound production was examined under different temperature 30, 37 and 42.
The incipient pH values of MRS broth were accommodated to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 with concentrated HCl and NaOH. The strain AFS18 was cultivated at 37â„ƒ for 48h (Prema, 2010).
The flasks were incubated at 37â„ƒ, as still cultures in 100ml anaerobic flasks sealed with butyl rubber membranes, in 100ml Erlenmeyer flasks sealed with cotton on a rotary shaker(100rpm) or cotton plugs in a still cultivated condition (Magnusson, 2001).
After incubation, the samples were collected for the determination of pH, inhibition activities and OD values.
2.7 Effects of temperature, pH and proteolytic enzymes on antifungal activity
The antifungal activity of the culture supernatants after the treatment of temperatures, different pH values or proteolytic enzymes was determined with the agar well diffusion assay.
The culture supernatants were divided into four samples and they were heated to 50, 70, 100, and 121 for 10 min, respectively. After cooling them down, the antifungal activity of each sample was detected.
The pH values of the cultures supernatants were adjusted to 2.5, 3.0, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with concentrated HCl and NaOH. The MRS broth adjusted to the same pH values served as a control.
The culture supernatants were treated with trypsin or pepsin (BioDee BioTech Co.,China). Samples were adjusted to the optimum pH values for each enzyme. i.e., 7.6, 2.0 for trypsin, pepsin respectively (Magnusson, 2001). After adjustment of the pH, the supernatants were treated with 100 µg of the respective enzyme per ml and incubated at 37°C for 1 h. Before evaluating the antifungal activity the pH of the supernatants was readjusted to the initial pH value 3.8. The MRS broth without treatment of proteolytic enzymes but modulated with the same pH values were used as control.
2.8 Identification of strain AST18.
LAB AST18 was identified from the 16SrDNA gene sequence. For 16S ribosomal DNA sequence determination, chromosomal DNA was isolated using the TIANamp Bacteria DNA kit (Tiangen Biotech Co., China). PCR amplification (TaKaRa, Japan) (94°C for 60 s, 55°C for 60 s, and 72°C for 90 s, 30 cycles) using primers 16S.S (5_-GGTGTAGCGGGTGAAATGCGAA-3_) and 16S.R (5_-CAGCCTACAATCCGAGCTGAG -3_) amplified approximately 700 bp of the 16S ribosomal DNA (Junguo Wang,2006 ). Sequencing of the amplified DNA was performed using the Thermo Sequenase dye terminator cycle sequencing kit (Tiangen Biotech Co., China) and the Applied Biosystems 3730xl DNA Analyzer (ABI, USA).
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3.1 Identification of LAB AST18.
The sequence was examined by comparing the obtained sequence in the NCBI database. The obtained sequences (continuous stretches of 611 bp) were aligned and clustered with sequences from the NCBI database. The 16S rDNA sequence of strain AST18 and Lactobacillus casei ATCC 334 were 99% homological. The strain AST18 was indentified as Lactobacillus casei AST18.
3.2 Screening of LAB for antifungal activity
A total of 60 LAB strains were screened for inhibitory activities towards Penicillium sp. and momur sp. After 72 hours of incubation in the MRS broth, all the 60 LAB culture supernatants had a pH between 3.5 and 4.0. 7 out of 60 showed certain inhibitory activities towards both molds. There's no inhibition observed towards two corruption yeast isolated from the spoiled cheese and 7 Saccharomyces cerevisiaes preserved in our laboratory. The sterile MRS broth adjusted to the pH value of 3.5 with concentrated HCl did not show any inhibition activities.
3.3 Influence of phenyl lactic acid, lactic acid and acetic acid on the growth of Penicillium.sp
Phenyllactic acid has already been known as a novel and effective organic acid produced by some strains of LAB. It has a broad spectrum activity against bacteria and fungal pathogens (Schnürer and Magnusson, 2005). The PLA concentration of the 7 LAB strains was analyzed by HPLC. All of the 7 strains can produce PLA. Sy03 produces the highest concentration of PLA which can reach to 16.24mg/L and A4 has the lowest production of 6.86 mg/L. But there are no relativity observed between the inhibition activity and the production of PLA (Fig1). That shows PLA is not the only antifungal compound in the culture supernatants.
The pH of the 7 strains is between 3.77 and 3.92 (Table 1). In order to determine the inhibition effect of lactic acid and acetic acid the pH values of MRS broth with lactic acid or acetic acid were adjusted to 3.77 respectively and there's no inhibition activity observed. So the lactic acid and the acetic acid are not effective on the antifungal activity individually.
3.4 Influence of cultivation temperature, incipient pH and aeration on antifungal compounds production of strain AST18
The optimum temperature was observed at 37â„ƒ. The growth of strain AST18 was inhibited when the temperature reached 42â„ƒ(Table 2).
The incipient pH values of MRS broth were accommodated to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 with concentrated HCl and NaOH. The strain AST18 growed well when the pH is between 6.0 and 6.5 and got the highest inhibition activity (Fig 2).
The flasks were incubated at 37â„ƒin different aerated conditions. There's a minor difference between cultures incubated as still cultures in cotton plug flasks and those incubated with air access on a rotary shaker. But the one with windtight plug showed less inhibition activities and OD values (date not shown).
The OD value of AST18 reached the stationary phase after 48h when it was cultivated in MRS broth at 37â„ƒ. The pH reached the stationary phase at 12h, but the highest inhibition activity observed after cultivation of 48h (Fig 3). This revealed the antifungal activity was not just relay on the pH values.
3.5 Effects of temperature, pH and proteolytic enzymes on antifungal activity
The activity was partly lost during heat treatment and there's no inhibition activity observed after the treatment of 121â„ƒ,10min(Table 3). Maximum activity was observed at pH values between 2.5 and 4.0. But it decreased rapidly between 4.0 and 7.0, and totally lost at higher pH values (Fig 4). This indicated that either organic acids or other pH-dependent antifungal compounds were responsible for the antifungal effect. The antifungal activity was not sensitive towards trypsin and pepsin (date not shown).
The antifungal activitives of 60 LAB strains were investigated. 7 out of 60 strains have the inhibitory activity towards penicillium sp. and Mucor sp. And there's no inhibition activities observed towards Saccharomyces cerevisiaes.
Gourama(1997)have shown that lactic acid was not inhibitory to fungi while the acetic acid concentration seemed to be more strictly related to the antifungal activity. Prema(2010)showed that phenyl lactic acid was inhibitory to fungi, but the acetic acid concentration seemed to be less related to the antifungal activity. This research showed all of the 7 strains which have antifungal activity can produce PLA, but the production was limited. The highest production of PLA was 16.24mg/L.The minimum production was 6.86mg/L. There's no strict dependability observed between the production of PLA and the antifungal activity. Phenyllactic acid was able to inhibite the growth of P.expansum IDM/FS2, A.niger FTDC3227 and IDM1, A. flavus FTDC3226 and F. graminearum IDM623 at a concentration of about 50 mg/ml (Lavermicocca et al., 2000). Prema (2010) detected that the minimum inhibitory concentration values against fungal cultures were ranged from 6.5 to 12.0 mg/ml for commercial 3-phenyllactic acid. The PLA production in this study was far below 6.5 mg/ ml. It indicated that there must be other pH dependant antifungal compounds in the culture supernatants. There's no antifungal activities observed in MRS broth which the pH was adjusted to 3.88 with lactic acid and hydrochloric acid separately. Organic acids can only penetrate the microbial cell wall in their undissociated form, The pKa of lactic, acetic, 3-phenyl-Llactic and caproic acid is 3.8, 4.7, 3.5 and 4.9, respectively (Cassandra, 2004). There's 36% antifungal activities lost when the pH ranged from 3.0 to 5.0. This suggested that organic acid were partly responsible for the antifungal activities. There must be other compounds related to the antifungal activities.
Lactic acid bacteria and their metabolites to inhibit the growth of mold and mycotoxin accumulation appears to be a promising biocontrol strategy in perishable foods or feed frequently contaminated by toxigenic fungal strains (Dalié, 2010). Dal Bello (2007) used the antifungal strain Lactobacillus plantarum FST 1.7 which can affect the growth of F. culmorum and F. graminearum to improve the shelf-life of bread. The Lactobacillus casei AST18 has the potential to use as protective culture in the preservation of yogurt, cheese and other food products. The fermentation properties of Lactobacillus casei AST18 is undereserach.
The results of the present study indicated that the antifungal effect of these strains could not simply be assigned to the low pH or organic acids but it has been suggested that they produced a wide spectrum of compounds that act synergistically towards corruption fungi. The further work is aimed in the purification of the antifungal compounds and the application of the Lactobacillus casei AST18 used as protective culture in the fermented dairy products.