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Isolation, In Vitro Probiotic Characterization of Lactobacillus plantarum and its Role on Italian ryegrass Silage Quality Enhancement.

Byline: Srisesharam Srigopalram, Hyung Soo Park, Soundharrajan Ilavenil, Da Hye Kim, Mariadhas Valan Arasu, Palaniselvam Kuppusamy, Kyung Dong Lee and Ki Choon Choi


Italian ryegrass (IRG) is an important forage type for feeding ruminants. IRG has difficult to ensile with good quality. Hence the inoculation IRG with lactic acid producing bacterial strains (LAB) gives an alternate solution to produce quality silage. Accordingly new Lactobacillus sp. KCC-32 was isolated from fermented animal manure. Biochemical and physiological studies showed that the strain belonged to Gram positive, produced gas from glucose and catalase-negative. The 16S rRNA sequence analysis revealed that KCC-32 showed 99% similarity towards Lactobacillus plantarum sp. Further, KCC-32 displayed potential probiotic characteristics including resistant to low pH, bile salt tolerance, auto-aggregation and hydrophobicity. The homo fermentative activity of KCC-32 resulted in the enhancement of Italian ryegrass silage quality.

In addition, KCC-32 added silage group showed significantly (P[?]0.05) increased lactic acid production (4.891 DM%) and the nutrient profile resulted with high crude protein content with less acid detergent fiber (ADF) and neutral detergent fiber (NDF) percentage when compared to control group. Further, the microbial count of KCC-32 silage group displayed significantly (P[?]0.05) high LAB count (24.3x107 cfu/g) and no fungal as well as yeast growth. Hence, this study suggests that KCC-32 has potential probiotic characteristics and the addition of KCC-32 to the IRG silage can improve the fermentation quality for the production of high-quality silage.

Keywords: Antifungal; Lactic acid; Silage; ADF; NDF; Probiotic


Probiotics are the viable microorganism that provides beneficial effects on the host health by maintaining and improving the intestinal microbial growth (Linda et al., 2015). Such probiotics possess various physiological functions such as assisting digestion, inhibition of pathogens, and antitumor activity (Clarke et al., 2012). The potential probiotic strain contains resistance to gastric stress and bile salt environments present in the gastrointestinal tract, more adhesion properties into the epithelial cells, and control pathogen adhesion using specific competition (Argyri et al., 2013; Hill et al., 2014). Further, probiotic can act as an antimicrobial agent, and various probiotic strains are reported to enhance the immune response activity of natural killer cells (Mera et al., 2012).

Probiotic strains are identified as safe because of their extended use in fermented food. Probiotic bacteria help food fermentation by various ways including (i) preservation by lactic acid production which act as antimicrobial agent (Fraqueza, 2015); (ii) The production of flavor compound that will provide the organoleptic properties (Smid and Kleerebezem, 2014); (iii) Improvement of nutritional value of the food; (iv) production of therapeutic agent and control of serum cholesterol (Nuraida, 2015). Adhesion of the probiotic strain to intestinal mucosa is considered as the main selection principle for probiotics (Ramos et al., 2013) as it sustains in the intestine for the long period and thus provides beneficial effects longer to the host (Mehmet et al., 2015). Among various probiotic strains, Lactobacillus plantarum has a significant role in food fermentation and health promoting properties.

Ensilation is the process that used for preserving forage crops. Silages are preserved forage crops that are preserved using ensilation process in many countries including Korea, China and Japan. During ensilation the inhibition of undesirable microbial growth mainly depends on sufficient organic acid production (Cai et al., 1999).

To achieve high organic acid production, homo and hetero fermentative lactic acid bacteria (LAB) are used as additives. In this process LAB change the carbohydrates into organic acids. Lactic acid is the main organic acid that is produced during silage fermentation (McDonald et al., 1991).

Lactic acid is responsible for the acidic nature of silage and inhibits the growth of undesirable microorganisms and plant enzymatic activities. Among homo and heterolytic fermentation, homolytic fermentation is considered more desirable because of high recovery of dry matter and energy. Lactobacillus sp. plays significant role in homolytic fermentation. Further, the Lactobacillus sp. mainly used in the fermentation of dairy products and preservation (Giraffa et al., 2010). Lactobacillus strains have been used as starters in the protection of fermented vegetables, dairy foods and fish for decades. Thus adding potential Lactobacillus inoculants to silage increasing the fermentation and thereby producing high quality silage (Avila et al., 2010). On the other hand, adding Lactobacillus to silage has numerous benefits including non-corrosive to farm machinery, less cost than enzyme formulation and do not pollute the environment (Weinberg et al., 2003).

Hence, the objective of this study has related to determine the in vitro probiotic characteristic of KCC-32 and its role in the enhancement of IRG silage quality.

Materials and Methods

Sample Collection, Biochemical Characterization and Anti-fungal Analysis

The animal manure samples were collected from Cheonan, South Korea. One gram of the sample was serially diluted using sterile water. Based on antifungal results, an effective strain was selected for further characterization and the strain was named as KCC-32. An overnight culture of KCC-32 was used for the analysis of biochemical and physiological properties. Megazyme assay kit (Bray, Co. WIcklow, Ireland) was used in the quantification of fermentative acid. The API 50 CH and API-ZYM kits (Marcy-I' Etoile, France) were used for the analysis of carbohydrate fermentation and enzyme production. The screening of antibiotic sensitivity was tested by disc diffusion method (Valan Arasu et al., 2013).

16s rRNA Sequencing and Gene Bank Deposition

The 16s rRNA gene sequencing was carried out at the Solgent Co (Seoul, South Korea) by the method of (Sanger et al., 1977). The genomic DNA of KCC-32 was isolated and purified by QIAquick(r) kit (Qiagen Ltd., Crawley, UK). The amplicons were sequenced using universal primers 27 F (5' AGA GTT TGA TCG TGG CTC AG 3') and the 1492 R (3' GCT TAC CTT GTT ACG ACT T 5'). The aligned 16srRNA sequence of the KCC-32 was subjected to BLAST with the non-redundant database of the NCBI GENE BANK. Further, the obtained 16s rRNA sequence of the isolate KCC-32 was deposited into NCBI Genebank.

Experiment on Low pH and Bile Salt Tolerance of KCC-32

The low pH tolerance and gastric juice resistance of KCC-32 were analyzed using the protocol described by (Charteris et al., 1998). For bile resistant analysis, fresh culture of KCC-32 was inoculated into the sterilized MRS broth containing bile salts such as 0.5% sodium deoxycholate and 0.3% oxgall (DCA Sigma, St Louis, MO, USA) and incubated at 37degC. 200 uL of the samples were taken after 24 h and 48 h respectively. Optical intensity was read at 600 nm.Without bile salts treated as control.

Auto Aggregation and Hydrophobicity Analysis of KCC-32

The aggregation (Del Re et al., 2000) and hydrophobicity (Rosenberg et al., 1980) were measured.

Sample Collection and Silage Making

Early stage of Italian ryegrass IRG was collected at, NIAS- RDA, Jeju. Two sets of 200 g of IRG were weighed. One set was served as control and the other was inoculated with (1.5x1010 cfu/g) KCC-32. Subsequently, the air inside the silage bag was removed and sealed. Similarly, triplicate samples were prepared and stored in room temperature for 45 days. After 45 days, the bags were opened, silage nutrient profile, and microbial content were analyzed.

Silage Nutritive Profile Analysis

The silage nutritive profile such as crude protein (CP: AOAC, 1990), Neutral detergent fibre (NDF: Van Soest et al., 1993), Acid detergent fibre (ADF: Van Soest et al., 1993), and Total digestible nutrient (TDN: Holland et al., 1990; Seo et al., 2010) were estimated using standard protocols.

Microbial Profile Analyzes

Serial dilution was made according to the method described by (Miller and Wolin, 1974). 100 uL of sample was spread on MRS specific media for Lactic acid bacteria and incubated at 28+-1degC for 48 h. 3 M petriflim (3 M Microbiology products, St. Paul, USA) was used for the counting of yeasts and molds. Potato dextrose agar (Difco) was used for the fungal population counting.

Estimation of Organic Acids

About 10 g of silage sample after 45 days of incubation was mixed with 90 mL distilled water and stored at 4degC for 24 h.

Firstly, samples were filtered through the filter paper (Whatman No. 6) and the filtrate was re-filtered using 0.22 um syringe filter before injection of samples in HPLC (HP1100., Agilent USA). The combination electrode was used to calculate the pH of the sample. The filtrate was stabilized with 5% meta-phosphoric acid and the filtrate was stored at -70degC. The organic acids such as lactic acid (HPLC), and acetic acid, butyric acid (Gas chromatography; GC-450, Varian Co., USA) were analyzed (Kristensen et al., 2007).

Evaluation of Statistics

All samples were evaluated in three replicates. SPSS/PC (v. 12.0) package was used for the analysis of variance of all the variables. The Duncan's multiple range test were used to determine the treatment mean difference at 5% probability level.


Isolation and Characterization of KCC-32

In this study, 15 LAB strains were isolated from animal manure. Among these isolated strains, single strain showed potential result in preliminary tests. Then the strain was named as KCC-32. Hence, the strain has been subjected to further experimental analysis. The 16 srRNA sequencing of the selected strain was blast with NCBI nucleotide sequence database. The results demonstrated more similarity with Lactobacillus plantarum ([greater than or equal to]99% similarity). Hence, we confirmed that KCC-32 belonged to Lactobacillus plantarum. The 16srRNA sequence of KCC-32 was assigned accession number KP091748.1 at NCBI Genebank.

Physiological and Biochemical Characterization of KCC-32

The isolated Lactobacillus plantarum KCC-32 was subjected to various biochemical and probiotic experiments. KCC-32 was grown in MRS broth for 48 h. After 48 h of growth, the microscopical observation resulted that KCC-32 was creamy in color, rod-shaped and Gram positive. The carbohydrate utilizing property of KCC-32 showed the fermentation of various carbohydrates (Table 1). The enzyme production analysis of KCC-32 confirmed the production of different intra and extra cellular enzymes (Table 2). Also, KCC-32 showed susceptibility to the common antibiotics (Table 3). Estimation of fermentative product of KCC-32 resulted majorly with lactic acid (127.23 ug/mL), acetic acid and Succinic acid respectively (Table 4).

Resistant Analysis of L. plantarum KCC-32 against Low pH and Bile Salts

The ability of KCC-32 to grow in GI tract conditions like low pH, resistant to bile salts was analyzed. The results confirmed that KKC-32 can survive in the GI tract conditions like low pH (Fig. 1A), tolerance towards induced gastric juice stress (pH 2 and pH 3) condition (Fig. 1B). No live cells were noticed below pH 2. Subsequently, KCC-32 displayed potential resistance against the toxic bile salts such as oxgall (0.3%) and sodium deoxycholate (0.5%) (Fig. 1C).

Auto Aggregation and Cell Surface Hydrophobicity

Analysis of L. plantarum KCC-32

The auto-aggregation and hydrophobicity experiments were carried out to find the ability of KCC-32 to form colonies inside the gut. In this experiment, KCC-32 showed strong auto-aggregation activity and the rate of auto-aggregation increased with the increase in time (Fig. 2B). Further, the cell surface hydrophobicity experiment was carried out using chloroform and xylene. The results revealed that KCC-32 showed strong hydrophobicity towards xylene with 59.07% (Fig. 2A).

IRG Silage Quality and Nutrient Composition Analysis

The IRG silage nutrient parameters including ADF, CP, TDN and NDF in control and experimental group were studied. A notable improvement in crude protein level of KCC-32 treated group was noted when compared to control IRG silage group. Simultaneously there was moderate decrease in ADF and NDF was noted as compared to control group. But in case of total digestible nutrient (TDN), slight increase was noted in KCC-32 inoculated group as compared to normal control group (Table 5). The microbial population profile of control and experimental group was listed in (Table 6). Significant (P[?]5) increase in LAB population was noted in KCC-32 inoculated group as compared to control group. Further, no yeast and fungal growth was noted in the KCC-32 treated experimental group when compared to normal control group. The level of organic acids produced during IRG silage ensilation process is listed in (Table 7).

There was significant (p<0.05) increase in lactic acid production was noted in KCC-32 inoculated group as compared to control group. Further, slight increase in acetic acid level and no butyric acid production was noted in KCC-32 treated group as compared to control group.


Among various probiotic strains, L. plantarum attracted more researchers worldwide because of its unique application in clinical as well as industrial needs. The character of L. plantarum to adopt different environments and various beneficial applications makes L. plantarum especially interesting and challenging. The most striking feature of L. plantarum was the high potential to import and metabolise a large number of carbohydrates (Alena et al., 2015). Accordingly, in this study the L. plantarum KCC-32, fermented different carbohydrates. Further, KCC-32 secreted different kind of important enzymes like -Galactosidase, -Galactosidase, - Glucosidase, and -Glucosidase etc. -Glucosidase breakdown the starch molecule and it converts the glycosides in to aglycones. The above conversion is a significant probiotic property because aglycones are easily absorbed by intestine (Ilavenil et al., 2016).

Table 1: Biochemical characterization of isolated L. Plantarum KCC-32using API 50 CHB system

Name of the carbohydrates###KCC-32

























Esculinferric citrate###+






















Potassium gluconate###+

Potassium2-keto gluconate###-

potassium 5-keto gluconate###+

Table 2: Analysis of intra and extra cellular enzyme production from KCC-32

Extracellular enzymes###KCC-32

Alkaline phosphatase###++

Esterase (C4)###++

Esterase lipase (C8)###+++

Lipase (C14)###++

Leucine arylamidase###+++

Valine arylamidase###++

Cystine arylamidase###++



Acid phosphatase###+










Table 3: Antibiotic sensitivity analysis of KCC-32

Name of Antibiotics###Concentration (g)###KCC-32

Chloramphenicol (C)###50###S

Kanamycin (K)###30###S

Nitrofurantoin (NIT)###50###R

Tetracycline (TE)###100###S

Streptomycin (S)###25###S

Sulphafurazole (SF)###300###S

Colistin methane sulphonate (CL) 100###R

Dicloxacillin (D/C)###1###R

Ampicillin (AMP)###10###S

Amikacin (AK)###30###S

Gentamicin (GEN)###10###S

Cefoxitin (CX)###30###R

Cefalexin (CN)###30###S

Cefuroxime (CXM)###30###S

Co-Trimoxazole (COT)###25###R

Table 4: Fermentative acid quantification in spent Lactobacillus plantarum KCC-32

Acid###Concentration (ug/mL)




It is very crucial to check the antibiotic sensitivity of lactic acid bacterial strains before considering them for human and animal consumption (Fraqueza, 2015). In this experiment, L. plantarum KCC-32 showed sensitivity to most of the tested antibiotics, which confirm the nonpathogenic nature of KCC-32 strain. The fermentative products secreted by the lactic acid bacteria contain antimicrobial metabolites which act as competitive weapons between the microbes. These antimicrobial metabolite productions ignited significant interest among researchers for natural food preservation using lactic acid bacteria as bio perseverant (Coda et al., 2011). Subsequently, the estimation of fermentative acids resulted with a high amount of lactic acid production which connects the effective involvement of lactic acid in beneficial properties of KCC-32.

Table 5: Nutritive value of IRG silage according to inoculation of lactic acid bacteria

Treatment###CP1) (%)###ADF2) (%)###NDF3) (%) TDN4) (%)


L. plantarum; KCC-32###9.66###32.97###51.94###62.85

Table 6: Changes of microbes on IRG silage according to inoculation of lactic acid bacteria

Treatment###LAB (x107 CFU1) Yeast (x103###Fungi (x103 CFU

###/g)###CFU /g)###/g)


L. plantarum KCC-32###24.3a###0###0

Similarly, bile salt has been known to play a significant role in colonization and metabolic activity of microbes in the intestine (Leite et al., 2015). Bile salts can act as an antimicrobial agent against intestinal microflora. Therefore, it is important to evaluate the bile salt tolerance of probiotic strains (Fontana et al., 2013). KCC-32 showed resistant to both bile salts such as oxgall (0.3%) and sodium deoxycholate (0.5%), which agreement with the report of (Ilavenil et al., 2016).

Probiotic bacteria have many kinds of positive natural activities on the host that implies greater impact on host intestinal mucosal surface adherence, which useful for prolonged presence of probiotic strains in the gut. Mucus plays a significant role in the maintaining intestinal micro flora by promoting microbial binding, serving as a nutrient source, and matrix for bacterial proliferation (Ilavenil et al., 2015). Conversely, mucus can inhibit pathogenic bacterial adhesion to the epithelium (Bautista-Gallego et al., 2013). In this study, the hydrophobicity and auto aggregation experiments showed potential adhesion skills of L. plantarum KCC-32. Particularly, KCC-32 showed more hydrophobicity towards xylene when compared to chloroform and the rate of auto aggregation increased with increase in time. Hence, the results clearly confirmed the adhesion ability of KCC-32 towards intestinal environments.

In silage nutrient profile analysis the nutrient parameters such as ADF, CP, TDN and NDF play significant role in determining silage quality. Particularly, the increase in the CP level at animal feed indicates the increase in rumen protein degradation (Olmos Colmenero and Broderick, 2006). On the other hand addition of homo fermentative lactic acid bacteria in silage preparation could lead to decrease in protein breakdown (Merry et al., 2000). Accordingly, there is no significant changes were noted between control and KCC-32 inoculated silage groups.

Table 7: Changes of pH and organic acids on IRG according to inoculation of lactic acid bacteria

Treatment###pH###Lactic acid (DM1)%)###Acetic acid (DM%)###Butyric acid (DM%)###Fliegs score


L. plantarum; KCC-32###3.85b###4.891b###0.417###0.061b###88

Similarly the other nutrient parameters like ADF, NDF and TDN showed no significant changes in KCC-32 inoculated group when compared to control group. This result confirmed that the addition of KCC-32 in IRG silage increases the nutritive value of the silage and also agreed with (Avila et al., 2010).

The growth of undesirable microorganisms like fungi and yeast decrease the aerobic stability of the silage that increases the protein degradation and thereby increase the butyric acid production. The production of butyric acid in silage severely affects the flavor and quality of the silage (Jatkauskas and Vrotniakiene, 2004). Similarly no yeast and fungal growth were noted in KCC-32 inoculated group.

Further, Addition of KCC-32 majorly increased the lactic acid production and then acetic acid production. The production of high amount of lactic acid confirmed the metabolic conversion of glucose and fructose in IRG silage by L. plantarum KCC-32 and agreement with (McDonald et al., 1991). Also, Most of the silages are affected by aerobic deterioration during warm climates (Ashbell et al., 2002). The aerobic yeasts are responsible for the aerobic deterioration of the silage and this could be eliminated by the addition of LAB inoculants (Danner et al., 2003). Accordingly, significant growth of L. plantarum increased the lactic acid and acetic acid production in KCC-32 inoculated group. The production of such organic acids automatically decreased the pH of the silage and thereby inhibited the growth of yeast colonies (Ilavenil et al., 2016).

Hence, simultaneous production of lactic acid and acetic acid decreased the production of less significant end products like ammonia as well as volatile fatty acid that cause severe dry matter loss (Avila et al., 2012).


In this study the L. plantarum KCC-32 was isolated from fermented animal manure and characterized based on carbohydrate fermentation, physiological and 16S rRNA gene sequence analysis. The in vitro probiotic characteristic of KCC-32 confirmed its ability grow in gastro intestinal like condition. Further, inoculation of KCC-32 in IRG silage enhanced the quality and nutrient profile. Subsequently, KCC-32 produced high amount of lactic acid in MRS growth medium as well as silage preparation. The overall experimental result confirmed that KCC-32 could be used in quality animal feed preparation.


This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (Project title: Development of Quality Improvement and Standardization Technique for Low Moisture Storage Forage; Project No. "PJ010916012015" Rural Development Administration, Republic of Korea. This study was supported by (2014) Postdoctoral Fellowship Program of (National Institute of Animal Science), Rural Development Administration, Republic of Korea.


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Author:Srigopalram, Srisesharam; Park, Hyung Soo; Ilavenil, Soundharrajan; Kim, Da Hye; Arasu, Mariadhas Va
Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Feb 28, 2017
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