Food-spoilage moulds such as Aspergillus, Fusarium & Penicillium are mycotoxinogenic in nature & play an obvious role in the deterioration of the quality & hygiene of foodstuffs by constituting highly toxic metabolites known as mycotoxins.
Mycotoxin contamination is still a serious problem in many parts of the world. This poses a threat to both livestock & human health, which results in great worldwide economic losses each year. Diverse physical & chemical methods have been developed for controlling mycotoxin-contaminated food & feed, whereas very few of these techniques have been practically considered. Therefore, a potent method has to be employed to reduce the presence of mycotoxins. Furthermore, some food-spoilage moulds have been discovered to have the capability to resist chemical preservatives, for example sorbate. The reduction of such moulds in food production is now of primary importance & there is great interest in developing effective & safe method for this purpose. It is crucial to reduce food losses by controlling fungal contamination at all stages of food process chains.
Three main stages can be defined to group fungal contamination factors:(i) the field, where water, soil & air are natural fungal niches; (ii) raw materials—such as post-harvest crops, meats, & milk—where fungal occurrence is related to food management during harvest or collecting, transportation, storage, & packaging & (iii) during food processing while manufacturing dairy, bakery, dry-ripened & drink products. Meanwhile, natural preservatives could be an alternative due to disadvantages of pesticides/fungicides & the high cost of production of new chemicals. In addition, the biopreservative capacity of lactic acid bacteria (LAB) is gaining much attention, due to consumers’ dem& for reducing the use of chemical preservatives.
As a result of this, several researchers have exploited antagonistic compounds (such as organic acids, hydrogen peroxide & other antimicrobial compounds such as bacteriocins) produced by LAB which are able to control pathogenic microorganisms & undesirable moulds in food & feed. However, much still need to be done on the antifungal activity of LAB, due to the great dem in finding natural preservative agents that can prevent fungal growth in food & feed. Accordingly, this study evaluates the ability of lactobacillus as a biopreservative agent to prevent food-spoilage moulds both in vitro & in vivo.
CONTENTS
CHAPTER 1 : INTRODUCTION 7-9
CHAPTER 2 : REVIEW OF LITERATURE 12-18
2.1 INTRODUCTION
2.2 IMPORTANCE OF LACTOBACILLUS IN FOOD INDUSTRY
2.3 SUBSTRATE USED IN BIOPRESERVATION
2.4 YAKULT AS A SUBSTRATE
CHAPTER 3 : MATERIAL REQUIRED & METHODOLOGY
3.1 SAMPLE COLLECTION 20-22
3.2 ISOLATION OF LACTOBACILLUSCASEI FROM YAKULT DRINK
3.3 CULTURES FOR ANTIFUNGAL & ANTIBACTERIAL ACTIVITY
3.4 DETERMINATION OF ANTIFUNGAL ACTIVITY
3.5 DETERMINATION OF ANTIBACTERIAL ACTIVITY
3.6 APPLICATION OF LACTOBACILLUS AS A BIOPRESERVATIVE
CHAPTER 4 : RESULTS 24-30
4.1 ISOLATION OF LACTOBACILLUS CASEI FROM YAKULT DRINK
4.2 DETERMINATION OF ANTIFUNGAL ACTIVITY
4.3 DETERMINATION OF ANTIBACTERIAL ACTIVITY
4.4 APPLICATION OF LACTOBACILLUS AS A BIOPRESERVATIVE
CHAPTER 5: DISCUSSION 32-34
CHAPTER 6 : SUMMARY & CONCLUSION 35-37
CHAPTER 7 : REFERENCES 39-43
ABBREVIATION
Illustrations are not included in the reading sample
LIST OF TABLES
TABLE NO: 4.3 Zone of inhibition (in mm) against bacterial pathogens
TABLE NO: 4.4(A) C°Culture of Lactobacillus strains with pathogenic bacteria (growth in terms of optical density) at
TABLE NO: 4.4(B) C°Culture of Lactobacillus strains with pathogenic bacteria (growth in terms of optical density) at
LIST OF FIGURES
FIG NO: 4.1(A) Yakult probiotic drink 24
FIG NO: 4.1(B) Lactobacillus casei from Yakult 24
FIG NO: 4.2 Anti-mould action of Lactobacillus casei in co-cultivation method 25
FIG NO: 4.3 Zone of inhibition in bacterial pathogens . 26
FIG NO: 4.4 Growth of different moulds on tomato juice 27
4.4(A) A.altemata
4.4(B) A.aureus
4.4(C) S.cerevisiae
4.4(D) A.niger
FIG NO: 4.4(E) Optical densities of target strains at 4°C 29
FIG NO: 4.4(F) Optical densities of target strains at 370C 30
CHAPTER 1 INTRODUCTION
Introduction
Food-spoilage moulds such as Aspergillus, Fusarium & Penicillium are mycotoxinogenic in nature & play an obvious role in the deterioration of the quality & hygiene of foodstuffs by constituting highly toxic metabolites known as mycotoxins.
Mycotoxin contamination is still a serious problem in many parts of the world (Richard & Payne 2003). This poses a threat to both livest°Ck & human health, which results in great worldwide economic losses each year (Guan et al., 2008). Diverse physical & chemical methods have been developed for controlling mycotoxin-contaminated food & feed, whereas very few of these techniques have been practically considered (Biernasiak et al., 2006). Therefore, a potent method has to be employed to reduce the presence of mycotoxins. Furthermore, some foodspoilage moulds have been discovered to have the capability to resist chemical preservatives, for example sorbate (Nielsen & Boer 2004). The reduction of such moulds in food production is now of primary importance & there is great interest in developing effective & safe method for this purpose. It is crucial to reduce food losses by controlling fungal contamination at all stages of food pr°Cess chains. Three main stages can be defined to group fungal contamination factors: (i) the field, where water, soil & air are natural fungal niches; (ii) raw materials—such as postharvest crops, meats, & milk—where fungal °Ccurrence is related to food management during harvest or collecting, transportation, storage, & packaging (Pitt et al., 2009) & (iii) during food
pr°Cessing while manufacturing dairy, bakery, dry-ripened & drink products. Meanwhile, natural preservatives could be an alternative due to disadvantages of pesticides/fungicides & the high cost of production of new chemicals (Laitila et al., 2002). In addition, the biopreservative capacity of lactic acid bacteria (LAB) is gaining much attention, due to consumers’ dem& for reducing the use of chemical preservatives (Magnusson et al., 2003). As a result of this, several researchers have exploited antagonistic compounds (such as organic acids, hydrogen peroxide & other antimicrobial compounds such as bacteri°Cins) produced by LAB which are able to control pathogenic microorganisms & undesirable moulds in food & feed (Messens & De Vugst 2002; Pulido et al., 2005). However, much still need to be done on the antifungal activity of LAB, due to the great dem in finding natural preservative agents that can prevent fungal growth in food & feed. Accordingly, this study evaluates the ability of lactobacillus as a biopreservative agent to prevent food-spoilage moulds both in vitro & in vivo.
LAB play a significant role in preservation & fermentation pr°Cesses. Therefore, they are the predominant microbiota of fermented products (Angmo et al., 2016). These beneficial bacteria are among the most important groups of industrial microorganisms & have wide applications in the food, chemical, cosmetic & medicine industries (Hatti-kaul et al., 2018, Vosough et al., 2022). LAB show a large set of technological & functional properties, which could enhance microbial safety & improve the sensory attributes of various foods. LAB also produce various antimicrobial substances, including organic acids, bacteri°Cins, hydrogen peroxide (H2O2) & diacetyl, which prevent the growth of many foodborne pathogens & spoilage microorganisms (Moradi et al., 2020).
Bacteri°Cins
Bacteri°Cins are anti-microbial proteins & peptides produced by bacteria. Lactic acid bacteria (LAB) our common bacteri°Cin producers & widely found in fermented foods. Characteristics of bacteri°Cins are taken into consideration, these peptides are thought to be promising therapeutic agents for the control of microbial pathogens, including multidrug resistant pathogens. The bacteri°Cin producing LAB as starter cultures in fermented foods can not only promote the fermentation by producing organic acids but also preserves the food by inhibiting the growth of spoilage bacteria & pathogens. Bacteri°Cins produced by LAB have great potential as natural food preservatives.
AIMS & OBJECTIVES
Aims & Objectives
1. Isolation of lactobacillus from Yakult probiotic drink.
2. Determination of antifungal & antibacterial activity of the Lactobacillus casei strain.
3. Application of lactobacillus (as a bio-preservative) against moulds & bacteria using tomato juice.
CHAPTER 2 REVIEW OF LITERATURE
Review of literature
2.1 Introduction
Lactobacilli are Gram positive (+) bacteria, shaped as rods which belong to the group of LAB. They are natural habitants, rapidly colonising mammalian in mucosal membranes such as oral cavity, intestine & vagina. In general, they are found where rich carbohydrate sources are available such as plants & materials of plant origin for example sewage & fermenting or spoiled food. (Bernardeau et al., 2007)
Lactobacillus is a group of bacteria with diverse species having higher GC content ranging from 32 to 53 mol% . (Kononen et al., 2007).
Lactobacilli are comprised a group of multifunctional microbes with shelf life extending characters of food as well as improving human health in the form of probiotics.
Lactobacillus species commonly isolated from GI tract are Lactobacillus acidophilus, L. brevis, L. plantarum, Lactobacillus salivarius & Lactobacillus fermentum. Lactobacillus are significantly required for gastrointestinal health & are not considered as a pathogen in healthy individuals except when having an ass°Ciation with dental caries. (Aguirre et al., 1993, Sharpe ME et al., 1973).
Lactobacilli distinctive ability is to produce lactic acid from carbohydrate sources, especially from lactose & glucose & many of them have been found to produce antimicrobial activity possessing molecules called bacteri°Cins. These compounds have gained major industrial interests due to their potential application to be used as natural preservatives (Rose, 1982, Board, 1983).
2.2 Importance of lactobacillus in food industry
Lactobacilli were the first microbes used by human beings for pr°Cessing foods & preservation of foods by inhibiting other microbial invasions that can cause food spoilage
& ultimately results in foodborne illness. (Adams MR. 1999, Konigs WN et al., 2000).
Lactobacillus as a starter culture
Nowadays, LAB are constantly used in the food pr°Cess industry in the form of starter cultures. Starter cultures, are carefully selected & propagated cultures of known strains of bacteria or yeasts in order to produce the suitable type of fermentation (homolactic, heterolactic, citrate etc.) .The starter cultures either consist of one pure strain of bacteria or yeasts or of a combination of strains of different microbial species .(Ross et al., 2005).
Application of Lactobacilli in Dairy Industry
The most commonly used species in dairy products are L.casei, L.helveticus, L.rhamnosus, L.lactis, L.curvatus & L.plantarum. Furthermore, Lactobacilli are incorporated into yogurt, cheese & fermented milk as probiotics due to their beneficial effect especially on acute & chronic inflammations of the gastrointestinal tract. (Bernardeau et al., 2007) In addition, due to the production of bacteri°Cins Lactobacilli also help on the preservation of dairy products. (Chen & Hoover, 2003).
Lactobacillus as Adjunct cultures
Secondary cultures, or adjunct cultures or adjuncts, are defined as any cultures that are deliberately added at some point of the manufacture of fermented foods, but whose primary role is not acid production. Adjunct cultures are used in cheese manufacture to balance some of the biodiversity removed by pasteurisation, improved hygiene & the addition of defined-strain starter culture. These are mainly non-starter LAB which have a significant impact on flavour & accelerate the maturation pr°Cess. (Cogan TM et al., 2007, Rattanachaikunsopon et al., 2010).
Application of Lactobacilli on Wine Industry
Lactobacilli are also applied in wine industry both for grape & fruit wines, such as cider. The organic acids existing in wine which are mainly malic & tartaric acid can be easily metabolised by Lactobacilli. (Board, 1983) Malic acid is converted to lactic acid & carbon dioxide, this phenomenon is called malolactic fermentation which is extensively used for fruit wines maturation. (Liu et al., 2003).
Application of Lactobacilli on non-beverage food products of plant origin
Lactobacilli are applied in the fermentation of sauerkraut that is the product of fresh cabbage. The starter culture for sauerkraut production is the normal flora of cabbage, in addition with L.plantarum & an amount of NaCl so to avoid the growth of
pathogenic bacteria. Another fermented product where Lactobacilli are involved is pickles.
Research on Bio-preservative activities of Lactobacillus plantarum strains in fermenting Cassava ‘fufu’ concluded that fermentation pr°Cess which involved L.plantarum caused a reduction in the level of pathogens. Twelve lactic acid bacteria strains were isolated during cassava fermentation. The antagonistic effect of Lactobacillus plantarum was determined by the agar diffusion method on S.typhii, Staphyl°C°Ccus Aureus & E.coli. Research showed that all the pathogens were inhibited by L.plantarum strains where Staphyl°C°Ccus aureus showed highest inhibitory zone followed by E.coli & S.typhii. Fermentation pr°Cess with the addition of Lactobacillus plantarum can give assurance of the control of pathogenic microorganisms by producing antimicrobial metabolites. (Obadina et al., 2006).
The Bio-preservative potential of marine Lactobacillus spp was studied. The lactic acid bacteria was isolated from fish gut & prawn muscles. A total of 116 strains were isolated from fish gut & prawn muscles, out of which only 10 strains were identified as potent strains for bacteri°Cin production. They are LAB1 Corynebacterium bovis, LAB2 Corynebacterium xerosis, LAB3 Lactobacillus alimentarius, LAB4 Lactobacillus animalis, LAB5 Lactobacillus casei, LAB6 Lactobacillus fermentum, LAB7 Lactobacillus plantarum, LAB8 Micr°C°Ccus varians, LAB9 Staphyl°C°Ccus epidermidiis, & LAB10 Strept°C°Ccus mitis. The lab strains were tested against 10 different antibiotics. The antibacterial activity of Lactobacillus strains was evaluated against 10 bacterial pathogens by well diffusion method. The study revealed that Lactobacillus strains of marine origin have the potential to be used as a bio-preservative especially in seafoods. (KJndira etal., 2011).
The study on Antimicrobial activity of Lactobacillus acidophilus that carry the bacteri°Cin gene was conducted. A total of 6 Lactobacillus acidophilus were isolated from 22 yoghurt samples. The antimicrobial activity was detected against different bacterial pathogens ie
E.coli, Pseudomonas aerogenosa, Aeromonas hydrophila, Proteus vulgaris, Staphyl°C°Ccus aureas, Staphyl°C°Ccus epidermidis, Strept°C°Ccus pyrogenes, & Bacillus subtilis. The study revealed that the six Lactobacillus acidophilus can produce antimicrobial compounds which were similar to bacteri°Cins & the isolates possess bacteri°Cin gene. (Anwar A. Abdulla., 2014).
The Bio control efficacy of Lactobacillus fermentum YML014 against food spoilage moulds using the tomato puree model was evaluated. Lactobacillus fermentum YML014 was isolated from Nigerian fermented food “Cassava”. The Antifungal activity of YML014 was investigated against A.flavus, A.niger & P.expansum by the co-cultivation method. The biopreservative potential of YML014 was also evaluated using tomato puree against P.exapnsum which resulted in an increase in the shelf life of tomato puree & act as a biopreservative. (Elizabeth O.Adedokun et al., 2015).
The study was undertaken to assess the Antibacterial effects of Lactobacillus isolates of curd & human milk origin against food-borne & human pathogens. Total 31 different lactobacilli (20 from curd & 11 from human milk) were identified. These strains belonged to five species Lactobacillus casei, L.delbrueckii, L.fermentum, L.plantarum & L.pentosus. The antibacterial activity of Lactobacillus strains were determined against 10 pathogenic strains ie Bacillus cereus, Listeria mon°Cytogenes, Staphyl°C°Ccus aureus, Strept°C°Ccus mutans, Salmonella enterica serovar Typhi, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Proteus mirabilis & Klebsiella pneumoniae. The antibacterial activities of cell free supernatants of all lactobacillus strains were determined through standard agar well diffusion assay. (Chetan Sharma et al., 2017).
The research on Antimicrobial activity of some Lactobacillus species against intestinal
pathogenic bacteria was conducted. The antimicrobial activity of four Lactobacillus species (Lactobacillus bulgaricus (PTCC 1332), Lactobacillus casei (PTCC 1608), Lactobacillus plantarum (PTCC 1058) & Lactobacillus Fermentum (PTCC 1638)) against four intestinal 15
pathogenic bacteria (Escherichia coli, Staphyl°C°Ccus aureus, Shigella dysenteriae & Salmonella paratyphi A). The study showed that the growth of all intestinal pathogens stopped in the presence of all Lactobacillus strains & they can be further used for human health benefits. (Hamid Tebyanian et al., 2017).
Study on the Characterization, yield optimization, scale up & biopreservative potential of fermencin SA715, a novel bacteri°Cin from Lactobacillus fermentum GA715 of goat milk origin was conducted. Fermencin SA715 is a novel, broad spectrum, non-porefirming & cell well ass°Ciated bacteri°Cin isolated from L.fermentum GA715 from goat milk. Antibacterial spectrum of fermencin SA715 was observed on the basis zone of inhibition against 16 indicator pathogens. Fermencin SA715 also doubled the shelf life & improved the microbiological safety of banana. (Samson Baranzan Wayah & Koshy Philip, 2018).
Isolation & characterization of Lactic acid bacteria isolated from fermented food of North-West himalayas. In this study, a total of 9 isolates was isolated from one of the traditional fermented products of North-West Himalaya region “Kaladhi”. The antagonistic activity of probiotic strains were observed against four pathogens ie Klebsiella pneumoniae, Enter°C°Ccus faecalis, St L.paracasei subsp.Tolerans rept°C°Ccus dysgalactiae & Pseudomonas aeruginosa by disc diffusion method. The results were predicted on the basis of inhibition zones. (Heena Sharma & Jasveen Bajwa, 2021).
Research on Antimicrobial activity of probiotic Lactobacilli against some pathogenic bacteria was conducted. The Lactobacilli was isolated from the probiotic product “Vitalactic B”. Two strains Lactobacillus plantarum & Lactobacillus acidophilus were present in it. The strains were tested for their antimicrobial activity against some pathogenic bacteria ie Staphyl°C°Ccus aureus, Escherichia coli, Pseudomonas aeruginosa & Klebsiella pneumoniae by the disk
diffusion method. The result concluded that the strains showed antimicrobial activity (inhibition zone) against Staphyl°C°Ccus aureus, Escherichia coli, Pseudomonas aeruginosa & no activity against Klebsiella pneumoniae. (Radwan Rushdi & Maryam Rushdi, 2022).
Investigating the Probiotic properties & Antimicrobial activity of Lactic acid bacteria isolated from an Iranian fermented dairy product “kashk” Fifteen bacteria were isolated from “kashk” sample which was collected from two regions of Iran. Out of fifteen isolates, eight isolates were identified as lactic acid bacteria. These isolates were Enter°C°Ccus faecium KKP 3772 (KF1), Enter°C°Ccus faecium C1 (KF2), Pedi°C°Ccus pentosaceus H11 (KF3), Pedi°C°Ccus pentosaceus VNK-1 (KK4), Lact°C°Ccus lactis RSg (KK1), Enter°C°Ccus faecalis P190052 (KK2), Enter°C°Ccus mundtii CECT972T (KK3) & Lactiplantibacillus plantarum PM411 (KK5). Survival of isolates in simulated digestive system, bile salts tolerance, antibiotic susceptibility, cell surface hydrophobicity, auto aggregation assay & antibacterial activity of the isolates were investigated. The antibacterial activity was tested against four pathogens ie Escherichia coli ATCC 25922, Staphyl°C°Ccus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 & Salmonella typhimurium PTCC 1609. (Bahareh Saboori et al., 2022).
2.3 Substrates used in biopreservation.
Lactobacillus acidophilus was isolated from different yoghurts. The antimicrobial activity of lactobacillus acidophilus was identified. (Anwar A. Abdulla., 2014).
Lactobacillus was isolated from curd & human milk & antibacterial effects of Lactobacillus was observed against food borne & human pathogens. (Chetan Sharma et al., 2017).
Cassava ‘fufu’ was used as a substrate for the isolation of Lactobacillus plantarum & its potential application for bio-preservation was determined. (Obadina et al., 2006).
Fish gut & prawn muscles were used as a substrate for the isolation of marine Lactobacillus spp & its bio-preservative potential was studied. (K.Indira et al., 2011). “ Cassava” Nigerian fermented food was used as a substrate for the isolation of Lactobacillus fermentum YML014. Bi°Control efficacy of Lactobacillus fermentum 17
YML014 was determined against food spoilage moulds using the tomato puree model. (Elizabeth O.Adedokun et al., 2015).
Goat milk was used as a substrate for the production of bacteri°Cin fermencin SA715. Characterization, yield optimization, scale up & biopreservative potential of fermencin SA715 was estimated. (Samson Baranzan Wayah & Koshy Philip, 2018).
“ Kaladhi ” traditional fermented food of North-West himalayas was used as a substrate for the isolation & characterization of Lactic acid bacteria & the antagonistic activity was studied against some food borne pathogens. (Heena Sharma & Jasveen Bajwa, 2021).
A commercial probiotic product “Vitalactic B” was used to isolate Lactobacillus strains. The antimicrobial activity of probiotic Lactobacilli was tested against some pathogenic bacteria. (Radwan Rushdi & Maryam Rushdi, 2022).
An Iranian fermented dairy product “Kashk” was used as a substrate for investigating the Probiotic properties & antimicrobial activity of lactic acid bacteria. (Bahareh Saboori et al., 2022).
2.4 Yakult as a substrate.
Yakult is a Japanese sweetened probiotic milk beverage fermented with the bacteria strain Lactobacillus casei Shirota. Yakult’s ingredients are water, skimmed milk, glucose-fructose syrup, sucrose, natural flavors (citrus) & live Lactobacillus casei Shirota bacteria. The strain was originally classified as being Lactobacillus casei.
CHAPTER 3 METHODOLOGY
Methodology
3.1 Sample collection.
Yakult probiotic drink was collected as a sample from nearby store.
3.2 Isolation of Lactobacillus casei from Yakult.
Lactobacillus casei was isolated from yakult drink & maintained on De man Rogosa & Sharpe (MRS) agar & stored at 4°C.
3.3 Cultures for antifungal & antibacterial activity.
Pure culture of Moulds (Aspergillus flavus (DIBNS) , Aspergillus niger (MTCC281) , Penicillium chrysogenum (MTCC192) , R.oryzae (MTCC1987) , A.alternata (MTCC6572) , S.cerevisiae (DIBNS) , Mucor sp (MTCC414) , S.boulardii (DIBNS) & A.famigatus (MTCC9657) ) & Bacteria such as(E.coli (MTCC443) , S.aureus (MTCC96) , P.aeruginosa (MTCC424) ) was obtained from the Microbiology laboratory of Dolphin (PG) Institute.
3.4 Determination of antifungal activity
The antifungal activity was determined by co-cultivation method. Pure culture of all moulds were obtained by in°Culating them on PDA slant. After incubation for 2 days, the culture of all moulds were prepared by in°Culating them on Sabouraud Dextrose broth. Also make broth culture of L.casei which was obtained from Yakult drink. In°Culate the L.casei on MRS broth & incubate for 24 hrs. After obtaining the growth of all moulds &
L.casei in a broth, make the MRS broth for the determination of antifungal activity. Pour 10 ml of MRS broth in each test tube. In°Culate the fungus (each per test tube) in 10 ml MRS broth as a “control”. In°Culate 500 pl of fungal broth along with 500 pl of L.casei broth in 10ml MRS broth as a “test” in each test tube. Incubate all the tubes at 30°C for 7 days. After Incubation, fungal mycelia was collected on Whatman filter paper & dried at 70°C until constant weight. After drying, measure the weight of all the dry moulds.
Calculate the percentage of growth inhibition (GI) using the formula:
GI (%) = C0-CF/C0 x 100
Where, C0 is the weight of dry mycelia (control),
Cf is the weight of dry mycelia after inhibition by Lactobacillus casei. (Elizabeth O. Adedokun et al., 2015).
3.5 Determination of antibacterial activity.
The Lactobacilli casei was in°Culated in MRS broth & incubated overnight. In°Culate the pure culture of bacteria such as E.coli, P.eruginosa & S.aureus in BHI broth & incubate for 24 hrs. Next day, the culture Of L.casei was centrifuged at 6000 rpm for 10 mins & then supernatant was separated from the pellet. Supernatant was divided into 4 groups for treatment-:
Heat treatment (boiling for 10 mins).
Neutralization to ph-7 with NaOH.
Treatment with catalase (H2O2).
Control (no treatment).
After the treatment, the supernatant were filtered with the help of Whatmann filter paper. Spread different bacteria (each per plate) on the surface of NA plates & after drying make wells in the plates (4 wells in each plate). Now, transfer 100 ul of supernatant in each well. Incubate the plates for 24 hrs at 37°C . Observe the zone of inhibition & measure the diameter of zone of inhibition. (Bahareh Saboori et al., 2022.
3.6 Application of lactobacillus as a bio-preservative.
Biopreservative potential of lactobacillus for fungal strains
The bio-preservative activity of lactobacillus was assessed using tomato juice purchased from gr°Cery store. Twenty grams of tomato juice mixed with SDA was poured into sterile petri dishes. 900 pl of sterile distilled water containing 100 pl of broth culture of fungus (A.niger, A.flavus, A.altemata & S.cerevisiae) was spread on the surface of control sample plates. In addition, 100 pl of broth culture of fungus along with 9:1 (w/v) of Lactobacillus casei was spread on the test plates. The plates were incubated at 27°C. Observe the growth of fungus in both control & test plates. (Elizabeth O. Adedokun et al., 2015).
Biopreservative potential of lactobacillus for bacterial strains
The c°Culture of lactobacillus strains with pathogens was determined as the previously described method (M. S. Bin Masalam et al., 2018) (with slight modifications) The antibacterial activity of L.casei in tomato juice was determined. 10 ml of fresh tomato juice was poured in each test tube. After that 2 ml of crude Lactobacillus casei was added in each test tube. In addition, 2 ml of bacterial culture such as S.aureus, P.aeruginosa & E.coli were added in test samples. Incubate the tubes at 4°C & 37°C . Microbial load of each treatment was monitored at a time interval of 24 hrs, 48 hrs & 72 hrs by taking OD on UV-Visible spectrophotometer at 540 nm.
The colony count was observed during storage period of 0, 7, 14, 21 and 30 days by standard spread plate count method. MRS agar was used to enumerate lactic acid bacteria while nutrient agar was used to enumerate S.aureus, P.aeruginosa & E.coli.
CHAPTER 4 RESULTS
Results.
4.1 Isolation of Lactobacillus casei from Yakult.
Lactobacillus casei was isolated from the Yakult probiotic drink. It was spreaded on the surface of MRS agar . Morphology characteristics of L.casei were rod shaped, gram positive, non sporing, non motile, colonies was creamy white in colour & growth °Ccur at an incubation period of 48 hrs. (Shown in fig.4.1A & B)
Illustrations are not included in the reading sample
Fig-4.1(A) Yakult Probiotic drink
Illustrations are not included in the reading sample
Fig-4.1(B) Lactobacillus casei from Yakult (Gram positive Rods)
4.2 Determination of antifungal activity.
The antifungal activity was determined by the co-cultivation method. The antifungal activity of L. casei was investigated against Aspergillus flavus, Aspergillus niger, Penicillium chrysogenum, R.oryzae, A.alternata, S.cerevisiae, Mucor sp, S.boulardii & Afumigatus). The highest percentage of inhibition was observed against A.alternata (34%) followed by A.niger (25%), S.cerevisiae (16%) & A.flavus (11%). Whereas, L.casei showed no inhibition against Penicillium chrysogenum, R.oryzae, Mucor sp, S.boulardii & A.fumigatus. (Shown in fig. 4.2)
Illustrations are not included in the reading sample
Fig-4.2: Anti-mould action of Lactobacillus casei in co-cultivation method
4.3 Determination of antibacterial activity.
The Antibacterial activity of L.casei was determined against three bacterial pathogens such as E.coli, S.aureus & P.aeruginosa. The supernatant of L.casei was treated by four treatments :
1. In control, all pathogens showed same zone of inhibition ie 0.6 mm.
2. In the heat treatment, none of the pathogen showed zone of inhibition.
3. In the catalase treatment, E.coli & P.aeruginosa showed same diameter of zone of inhibition ie 0.6 mm. (Shown in table 4.3)
4. In the neutralization treatment, the highest zone of inhibition is shown by E.coli followed by S.aureus whereas P.aeruginosa showed no zone of inhibition. (Shown in fig 4.3D).
Illustrations are not included in the reading sample
Fig- 4.3 Zone of inhibition in bacterial pathogens
Table- 4.3: Zone of inhibition (in mm) against bacterial pathogens
Illustrations are not included in the reading sample
4.4 Application of Lactobacillus casei as a biopreservative.
Biopreservative potential of Lactobacillus for fungal stain.
The preservative effect of L.casei was shown with tomato juice. Delay of six days mould growth was seen in all the four fungal pathogens such as A.alternata, A.flavus , S,cervisiae & A.niger at 27°C compared to the control plates. Delay of nine days was only observed in S.cerevisiae treated with cells of L.casei compared with the control plates. (Shown in 4.4 A, B, C & D)
Illustrations are not included in the reading sample
Fig-4.4(D) A.niger
Fig-4.4 Growth of different moulds on tomato juice
Biopreservative potential of Lactobacillus for bacterial stain.
The biopreservative potential of L.casei was evaluated using tomato juice against three bacterial pathogens such as E.coli, S.aureus & P.aeruginosa at two different temperatures 4°C & 37°C.
At 4°C, the growth of pathogens was decreased on fifth day of incubation as compared to the third day. The minimum growth was shown by E.coli followed by P.aeruginosa followed by S.aureus. (Shown in table 4.4A & fig 4.4E)
At 37°C, the minimum growth was shown by E.coli followed by P.aeruginosa & maximum growth in S.aureus at fifth day. At 37°C, S.aureus showed maximum growth on fourth day as compared to other bacterial pathogens. (Shown in table 4.4B & fig 4.4F).
Table-4.4(A) : C°Culture of Lactobacillus strains with pathogenic bacteria (growth in terms of optical density) at 4°C
Illustrations are not included in the reading sample
Table-4.4 (B) : C°Culture of Lactobacillus strains with pathogenic bacteria (growth in terms of optical density) at 37°C
Illustrations are not included in the reading sample
Fig-4.4(E): Optical densities of target strains at 4°C
Illustrations are not included in the reading sample
Fig-4.4(F): Optical densities of target strains at 37
Illustrations are not included in the reading sample
CHAPTER 5 DISCUSSION
Discussion
In this study Lactobacillus casei was isolated from probiotic Yakult drink in MRS culture medium which is a selective medium for the lactobacillus spp isolation. The colony morphology on MRS agar medium was off white, round with smooth edges & rose from centre. On microscopic examination lactobacillus casei was rod in shaped, gram positive. The similar findings was reported by (Ali et al., 2010) as they isolated five lactic acid bacteria. All isolates were Gram-positive, motile, mesophilic, facultative anaerobic, catalase negative, rods with lactic acid production. The isolates have been characterized & identified by a polyphasic approach, combining with various phenotypic & genetic characteristics. Another study also showed that six isolates of Lactobacillus acidophilus were isolated from twenty two samples of yoghurt. (Anwar A. Abdulla., 2014). Indira et al., 2011 also isolated 116 strains of LAB were isolated from fish gut & prawn muscles, out of which 10 strains were identified as potent strains for bacteri°Cin production. . Nine isolates were isolated from fermented food of North-West himalayas “Kaladhi” (Heena Sharma & Jasveen Bajwa, 2021). Similarly, LAB were isolated from Iranian fermented dairy product “Kashk” Fifteen bacteria were isolated, out of which eight were identified as LAB. (Bahareh Saboori et al., 2022).
In this project we studied the antifungal & antibacterial activity of lactobacillus casei against moulds and bacteria . For the determination of antifungal activity, the growth inhibition percentage by Lactobacillus casei was evaluated against different moulds.
In the similar study, the antifungal activity of Lactobacillus fermentum YML014 against food spoilage moulds was studied. (Elizabeth O.Adedokun et al., 2015).
The antibacterial activity of Lactobacillus casei was studied by agar well diffusion method. In this method, the supernatant of L.casei was treated into four groups ie control, heat treatment, neutralization to ph-7 & treatment with catalase. The bacterial pathogens such as E.coli, S.aureus & P.aeruginosa were spreaded on NA plates. The treated supernatant was then poured into the wells & the zone of inhibition was observed against bacterial pathogens.
In the previous studies, the antibacterial effects of Lactobacillus was studied against 10 pathogenic bacterial strains. The antibacterial activity was determined through standard agar well diffusion assay. (Chetan Sharma et al., 2017).
The antibacterial spectrum of a bacteri°Cin Fermencin SA715 was observed on the basis of zone of inhibition against 16 indicator pathogens. (Samson Baranzan Wayah & Koshy Philip, 2018).
Similarly the antagonistic activity of 9 isolates of Lactic acid bacteria were observed against four pathogens by the disc diffusion method. (Heena Sharma & Jasveen Bajwa, 2021).
In this project, we studied the biopreservative potential of Lactobacillus casei against different moulds & bacteria using tomato juice. The study was conducted in two different methods. In the first method, the broth culture of fungus was spreaded on the tomato juice as control plates. In addition, the broth culture of L.casei along with fungal broth was spreaded on the surface of test plates. The growth was observed on particular days on both plates.
In the second method, the tomato juice was poured into the test tubes. The L.casei was in°Culated into the juice as control & in the test sample bacterial broth were also in°Culated along with L.casei. The microbial load was evaluated & OD was taken on UV-Visible spectrophotometer.
In the previous research on biopreservative activities of L.plantarum strains in Cassava ‘fufu’ resulted in the reduction of pathogen level. The effect was determined by agar well diffusion method on some bacterial pathogen. (Obadina et al., 2006).
Similarly, the biopreservative potential of marine Lactobacillus.spp was studied. The 10 Lactobacillus strain were identified & tested against 10 antibiotics & 10 bacterial pathogens. It concluded that marine Lactobacillus spp can be used as biopreservative in seafoods. (KJndira et al., 2011).
CHAPTER 6 SUMMARY & CONCLUSION
Summary
The use of natural preservatives may be effective as comparison to chemical preservative to maintain the quality of minimally pr°Cessed products by having an antimicrobial effect, by inhibiting spoilage and avoiding oxidative pr°Cesses. Moreover, the application of living antagonistic microorganisms can provide protection against pathogens by means of a combination of mechanisms including the production of antimicrobial compounds. This pr°Cess is known as bioprotection or biological control.
To minimize the spoilage of tomato juice and make them preserve for long period of time we did this research on biopreservation of tomato juice using lactobacillus casei isolated from the yakult and testing antimicrobial activity against pathogenic bacteria and fungi .Lactic acid bacteria group was chosen as a target for the selection of bi°Control agents for several reasons. Firstly, they are considered as GRAS, & fit all recommendations for use in food stuff. Secondly, LAB is part of the natural microbiota. Thirdly, LAB are known as good antagonists & have been successfully used as bi°Control agents of several food products.
Lactic acid bacteria (LAB) are known to preserve foods from spoilage, prolong the shelf life & provide better quality & safety of foods. Spoilage microorganism such as Botrytis cinerea, Fusarium, Rhiz°Ctonia solani, Erwinia carotovora, Xanthomonas, Penicillium, Staphyl°C°Ccus, Bacillus spp & Aspergillus spp play an undeniable role in the deterioration of the marketable quality of vegetables & constitutes major economic problem. Moreover, contamination by spoilage microorganisms can cause considerable economic losses to the fresh produce.
The first stage of study was the isolation of Lactobacillus casei from the probiotic drink Yakult. The pure culture of moulds & bacteria were obtained from the Microbiology laboratory of Dolphin (PG) Institute. The antifungal activity of Lactobacillus casei was determined by cocultivation method against nine fungal moulds. The percentage of growth inhibition of fungal moulds by Lactobacillus casei was evaluated.
Then, we tested the antibacterial activity of Lactobacillus casei against three bacterial pathogens such as E.coli, P.aeruginosa & S.aureus.. In this method, supernatant of L.casei was treated by four treatments & then poured into the wells. Then, the zone of inhibition was determined.
At last the biopreservative potential of Lactobacillus casei was studied using tomato juice. In the first method, the biopreservative potential of Lactobacillus casei was tested against four fungus such as A.niger, A.flavus, A.altemata & S.cerevisiae. In the second method, the biopreservative activity of Lactobacillus casei was evaluated against three bacterial pathogens such as S.aureus, P.aeruginosa & E.coli.
Conclusions
This work is designed to investigate the effectiveness of lactic acid bacteria metabolites in preserving tomato juice against fungal and bacterial pathogens.The major conclusions of the present project are as follows
- In this study , Lactobacillus was isolated on MRS agar from yakult probiotic drink & total 3 number bacterial isolates & total 9 number fungal isolates.
- L.casei showed maximum antifungal activity against A.alternata followed by A.niger, S.cerevisiae & A.flavus.
- The supernatant of L.casei showed different zone of inhibition in the four treatments against three bacterial pathogens ie E.coli, P.aeruginosa and S.aureus.
- The biopreservative potential of L.casei was seen maximum in S.cervisiae (delay of 9 days in mould growth was seen in tomato juice).
- At 4°C & 37°C, E.coli showed minimum growth & inhibited by L.casei in the tomato juice followed by P.aeruginosa & S.aureus.
- At 4°C Lactobacillus showed better inhibition activity than at 37°C.
Lactic Acid Bacteria strains could survive post harvest conditions & were effective against spoilage microorganisms. The selected Lactobacillus strain reduced the growth of most fungal & bacterial pathogens & worked as biopreservative in tomato juice. The efficacy of Lactobacillus casei was high against spoilage microorganisms & can be used as biopreservative.
The beneficial effects on these important microbiological characteristics revealed the potential of the Lactobacillus for application as a biopreservative and as a means for extending the storage life of fresh tomato juice.
CHAPTER 7 REFERENCES
References
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APPENDICES
APPENDIX I
(Culture Media)
Brain Heart Infusion (BHI) Broth
Illustrations are not included in the reading sample
MRS - Media (Man, De Rogosa & Sharpe)
Illustrations are not included in the reading sample
Nutrient agar
Illustrations are not included in the reading sample
Sabouraud Dextrose Agar (SDA) medium
Illustrations are not included in the reading sample
Sabouraud Dextrose Broth (SDB) medium
Illustrations are not included in the reading sample
APPENDIX - II
(Composition of stains)
Gram Stain Solutions
Frequently Asked Questions About the Language Preview
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This document is a language preview from a publishing company, designed for academic use in analyzing themes. It includes a title, table of contents, objectives and key themes, chapter summaries, and keywords.
What does the table of contents include?
The table of contents lists the following chapters: Introduction, Review of Literature, Material Required & Methodology, Results, Discussion, Summary & Conclusion, and References.
What does the Review of Literature cover?
The Review of Literature covers topics such as the introduction to *Lactobacillus*, its importance in the food industry, substrates used in biopreservation, and Yakult as a substrate.
What methodology is described in the document?
The document outlines methodologies for sample collection, isolation of *Lactobacillus casei* from Yakult drink, cultures for antifungal & antibacterial activity, determination of antifungal activity, determination of antibacterial activity, and application of *Lactobacillus* as a biopreservative.
What kind of results are presented?
The results include findings from the isolation of *Lactobacillus casei* from Yakult drink, determination of antifungal activity, determination of antibacterial activity, and application of *Lactobacillus* as a biopreservative.
What is the main focus of the introduction?
The introduction discusses food-spoilage molds, mycotoxin contamination, and the potential use of natural preservatives like lactic acid bacteria (LAB) as biopreservatives.
What are the aims & objectives outlined in the document?
The aims and objectives include the isolation of *Lactobacillus* from Yakult probiotic drink, determination of antifungal & antibacterial activity of the *Lactobacillus casei* strain, and application of *lactobacillus* as a bio-preservative against molds & bacteria using tomato juice.
What are some of the specific bacteria and fungi mentioned?
Specific microorganisms mentioned include *Aspergillus flavus, Aspergillus niger, Penicillium chrysogenum, R.oryzae, A.alternata, S.cerevisiae, Mucor sp, S.boulardii, A.famigatus, E.coli, S.aureus, P.aeruginosa*, and various *Lactobacillus* species such as *L.casei, L.acidophilus, L.plantarum*, among others.
What applications of Lactobacilli in the food industry are discussed?
Applications discussed include its use as a starter culture, in the dairy industry (yogurt, cheese, fermented milk), as adjunct cultures, in the wine industry, and in non-beverage food products of plant origin (sauerkraut, pickles).
What research studies are cited in the Review of Literature?
Numerous research studies are cited, covering topics like the biopreservative activities of *Lactobacillus plantarum* in fermenting Cassava, the potential of marine *Lactobacillus spp*, the antimicrobial activity of *Lactobacillus acidophilus*, the bi°Control efficacy of *Lactobacillus Fermentum*, antibacterial effects of *Lactobacillus* isolates, antimicrobial activity of *Lactobacillus* against intestinal pathogens, and more.
What substrates besides Yakult are mentioned for isolating Lactobacillus?
Other substrates mentioned include curd, human milk, Cassava ‘fufu’, fish gut, prawn muscles, Nigerian fermented food “Cassava”, goat milk, “Kaladhi” (traditional fermented food), and an Iranian fermented dairy product “Kashk”.
What methods are used to determine antifungal and antibacterial activity?
The determination of antifungal activity utilizes a co-cultivation method. Antibacterial activity is assessed through methods like agar well diffusion assays with various treatments of *Lactobacillus* supernatant (heat treatment, neutralization, catalase treatment).
How is the biopreservative potential of Lactobacillus assessed?
The biopreservative potential is assessed using tomato juice as a model, observing the growth of target microorganisms (fungi and bacteria) in the presence and absence of *Lactobacillus* at different temperatures (4°C and 37°C), and measuring optical densities (OD) over time.
What conclusions are drawn in the summary and conclusion?
The summary concludes that *Lactobacillus* strains can survive post-harvest conditions, are effective against spoilage microorganisms, can reduce the growth of fungal and bacterial pathogens, and work as biopreservatives in tomato juice. Specifically, *L.casei* shows better inhibition activity at 4°C than at 37°C. Overall *Lactobacillus casei* has the potential for application as a biopreservative and as a means for extending the storage life of fresh tomato juice.
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- Gauri Singh (Author), Ashok Singh (Author), Manu Sharma (Author), 2024, Bio Preservative Potential of Lactobacillus against Different Moulds and Bacteria Using Tomato Juice, Munich, GRIN Verlag, https://www.grin.com/document/1496185
