#ep 13 all pulp no juice

[18catsreading]

Get in, Loser, we're going to Rat World!

[irispublishersagriculture]

Iris Publishers - World Journal of Agriculture and Soil Science (WJASS)

Isolation, Identification and Characterization of Pectinase Producers from Agro Wastes (Citrus sinensis and Ananas comosus)

Authored by Ebuehi OAT

When fruit is detached from the living plants it cannot repair its cells or defend its tissues against natural deterioration. There are many reasons of fruit destruction, but main cause of fruit spoilage is invasion by microorganisms such as mould, yeast and bacteria. Fruits contain high levels of sugars and nutrients and their low pH make fruits particularly desirable to fungal decay [1]. During postharvest handling, mechanical damage causes the release of enzymes present in the cell tissues. These enzymes break down the cellular material. Pectinases are the first enzymes to be secreted by fungal pathogens when they attack plant cell walls, weaken the plant cell wall and expose other polymers to degradation by hemicellulases and cellulases [2]. Pectinases are one of the most widely distributed enzymes in bacteria, fungi and plants insects, nematodes, plants, and protozoan [3]. Fungi are considered to be prospective enzyme producing sources.

Pectinases breaks down pectin and are of significant importance in the current biotechnological era with their all-embracing applications in fruit juice extraction and its clarification, scouring of cotton, degumming of plant fibers, waste water treatment, vegetable oil extraction, tea and coffee fermentations, bleaching of paper, in poultry feed additives, in alcoholic beverages and food industries [4]. Pectinases constitute approximately 10% of the total enzyme production in the world market and 25% of global sale in the food industry [5].

 In the fruit juice industry, they are used for clarification of fruit juice by reduction in viscosity, increasing of juice yield by enzymatic liquefaction, and maceration of pulps, enhancing pigmentation by extracting more anthocyanin [6]. Microbial pectinases have been widely studied due to the requirement of highly productive strains and the cost-effective production of enzymes. In the present study, the pectinase was purified from a newly isolated Bacillus subtilis strain and this enzyme was characterized Identification and characterization of various pectinase producers have been published. There is paucity of information from the literature on combined action of two fruits and their mode of action. The aim of this study is to isolate and characterize based on primary and secondary screening, pectinase producers and how to optimize them from Agro-wastes, orange and pineapple

Materials and Methods

Reagents

The reagents and chemicals used in the study include: Pectin, K2HPO4, MgSO4.7H2O, NaCl, CaCl2.2H2O, FeCl3.6H2 O, yeast extract, Agar, urea, (NH4)2SO4, MgSO4, FeSO4, and glucose, were all of analytical grade and were purchased from Hi Media and Sigma Chemicals, USA.

Materials

Collection of samples

The fresh oranges were plucked from a tree in Alagomeji, Lagos, Nigeria. The fresh pineapples were gotten from a pineapple plantation in Epe, Lagos. The oranges and pineapples which were intended to be rotten were purchased from Mushin market, Lagos, Nigeria. The rottenness of the oranges and pineapples was obtained by leaving the purchased fruits for 14 days on a flat surface in the laboratory.

Isolation of pectinolytic fungi

Fungi were isolated from the rotten fruits (oranges and pineapples) by using the modified pectin agar medium. In brief, two-fold dilutions of the spoiled samples were plated in pectin agar medium and incubated at room temperature for 6 days. The strains grown in the plate were subcultured into pectin agar medium separately and flooded with 1% cetrimide for primary screening. The pectinolytic fungi produced a clearing zone when exposed to cetrimide [7].

Secondary screening

The fungal strains which showed clearing zones in pectin agar medium were selected for secondary screening by estimating the polygalacturonase (PG) and pectin lyase (PL) production under solid state fermentation by using the modified medium [8].

Isolation of pectinolytic bacteria

The isolates of both the rotten and fresh pineapple were cultured and incubated at 35°C for 48 h. Thereafter, the distinct bacteria colonies were streaked on nutrient agar and incubated at 35 °C for 24 h. When Gram-stained films were examined, the isolates that appeared as Gram-positive rods was identified as Bacillus subtilis based on result from biochemical test as enumerated [9-10]. The Bacillus isolates were maintained in nutrient agar slants at 4°C.

Enzyme assays

Polygalacturonase activity was measured according to the method described by [11-13] One unit of enzyme activity has been defined as the amount of enzyme that releases one μ mol of galacturonic acid /min under the assay conditions [12-13]. Pectin lyase activity was measured by the reaction between unsaturated end products of pectin degradation and thiobarbituric acid. One unit of activity is the amount of enzyme causing a change in absorbance of 0.01 under the conditions of the assay [14].

Effect of inoculum age, inoculum size, and incubation time

Inoculum age was optimized by inoculating the medium (secondary culture) with inocula (primary culture) of varying age, namely 8, 12, 16, 20, and 24 h, and assaying the filtered broth for enzyme activity. To study the effect of inoculum size, (103-106CFU/ ml) inoculum was used to inoculate the production medium, and polygalacturonase activity was assayed in the broth. The effect of incubation time was studied by incubating the microorganism in production medium for different time intervals (24, 48, 72 and 96 h) and measuring the enzyme activity.

Effect of temperature and pH

Most favorable production temperature was studied by incubating the production medium at different temperatures (20, 30, 35 and 40 °C). Polygalacturonase activity was assayed by using the standard method [11]. For optimizing the production of pH, the production medium varying pH, namely, 4.0, 4.5, 5.0, 5.5, was used for enzyme production, and activity was measured.

Effect of nitrogen source

The effect of various nitrogen sources (Ca (NO3)2, (NH4)2SO4, (NH4)2NO3, NH4Cl, NaNO3, KNO3, and Urea on the production of enzyme was studied by supplementing 0.1% w/v of these to the production media.

Effect of D-galacturonic acid on polygalacturonase production

To study the effect of D-galacturonic acid on polygalacturonase production, it was added to the culture broth at a final concentration of 0.3, 0.6, 0.9, 1.2, and 1.5%w/v under aseptic conditions. The resulting extracellular polygalacturonase activities produced were measured by estimating the reducing groups produced (Figures 1-6).

Discussion

Pectinolytic enzyme can be derived from different sources [15-17]. However, pectinase producing microorganisms have due advantage over other sources because they can be subjected to genetic and environmental manipulations to increase yield [18]. It has been reported that most Bacillus sp, enhances the production of pectinase [19]. This result indicates that orange and pineapple contain appreciable amount of pectin. The pectin was used as inducer for the production of pectinolytic enzymes. In this study, pectinase production by Bacillus subtilis and Aspergillus niger were compared. Our results show that both Bacillus subtilis and Aspergillus niger were able to produce pectinase. The isolates were later designated based on morphology as Bacillus subtilis, and Aspergillus niger are among the most studied pectinolytic fungi (Sukumaran et al., 2005; Favela-Torres et al., 2006). [20] also has reported that Bacillus sp, has produced higher yield of pectinase compared to most other microbes such as Aspergillus.

Temperature is very important factor for microbial growth as well as microbial product formation. The incubation temperature greatly affects the microbial growth rate, enzyme secretion, enzyme inhibition, and protein denaturation. Thermal stability and activity of pectinases are of great significance in biotechnological process. In this study, it was reported that 30 °C was the optimum incubation temperature for fresh and rotten orange and fresh pineapple but 35 °C was observed as the optimum incubation temperature for rotten pineapple. In previous report on A. niger, using orange and pineapple D-galacturonic acid as carbon source exhibited maximum activity at 35 °C which was in accordance to findings by [21].

The initial pH of the fermentation medium plays a vital role in determining the level of metabolite synthesis. The stability of the microbial metabolite is also dependent on the hydrogen ion concentration of the medium [22]. In this study it was found that the optimum pH condition for the production of pectinase is pH 5 for fresh and rotten orange and pineapple respectively. According to Spagna et al., 1996, maximum pectinase activity was under pH 5. The optimum pH from this study is comparable to pectinase of Penicillium varidictum and Penicillium oxalicum [23-25]. [26] reported that optimum pH 5.5 for pectinase activity from thermotolerant Aspergilus sp.

Maximum pectinase activity was observed with 104 CFU/ml and 103 CFU/ml inoculums size for both fresh and rotten oranges and pineapples respectively. The effect of inoculum sizes for the production of pectinase fresh and rotten pineapple respectively. Optimum inoculums density is an important consideration for fermentation process since accumulation of spore can inhibit growth and development of the culture organism [27].

An adequate supply of carbon as energy source is critical for optimum growth of organism and its metabolism. In the present study, supplementing carbon sources increased pectinase production on solid state. According to [28] low enzyme production with other carbon sources might be because of catabolite repression. Glucose is known to repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources; some of these genes are also repressed by other sugars such as galactose, [29].The effect of D-galacturonic acid with various concentrations (0.3%, 0.6%, 9%, 1.2%, 1.5%) was evaluated in this study and it was observed that there were increase in the pectinase activities with increase in the various concentrations of the D-glucoronic acids as [29] found that pectin and polygalacturonic acid promoted the production of pectic enzyme.

The effect of nitrogen sources was also observed in this study for both rotten and fresh oranges and pineapples. Of the various nitrogen sources used, maximum pectinase activity was observed when medium was supplemented with NH4Cl for the whole samples. Meanwhile, among the tested nitrogen sources, ammonium sulphate (NH4SO4) and ammonium nitrate (NH4NO3) increased the pectinase productivity on SSF though their effect was not significant. The result is in agreement with [30], who found that ammonium sulphate and ammonium nitrate were good nitrogen sources for pectic enzyme production from A. niger. Moreover, Sarvamangala et al., (2006) revealed that ammonium sulphate did influence production of pectinase positively in solid-state conditions

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[justlookatthosesausages]

About me ~ (part 1/4)

1. coffee mugs, teacups, wine glasses, water bottles, or soda cans? Mmmmh tea cups !

2. chocolate bars or lollipops? Lollipops! Even if I love chocolate, I actually don’t like chocolate bars that much

3. bubblegum or cotton candy? Bubblegum!

4. how did your elementary school teachers describe you? Extremely distracted, I used to have (and still have, in a way) a very high level of permanent daydreaming and sometimes I would miss like 30 full minutes of class – which is a problem when the lesson is like, important calculus lol –

5. do you prefer to drink soda from soda cans, soda bottles, plastic cups or glass cups? I actually hate soda! Any beverage with gas or anything that tickles the tongue like gas (i.e. alcohol) makes me shiver in disgust :(

6. pastel, boho, tomboy, preppy, goth, grunge, formal or sportswear? Where do I fit the ‘I own 40 video games/tv shows/movies references tshirts’ category in there lol

7. earbuds or headphones? The duality: I hate the sensation of earbuds, but as I wear glasses, headphones starts to hurt my temples after a while… Still picking headphones though lol

8. movies or tv shows? Definitely movies, I’m a huge cinema fan

9. favorite smell in the summer? Barbecue grillllllll

10. game you were best at in p.e.? (is that the American equivalent of EPS?) uhm I’d say Climbing?

11. what you have for breakfast on an average day? A glass of orange juice (with no pulp. I hate pulp. Yay picky eater me) and some chocolate biscuits :D

12. name of your favorite playlist? Calm (literally XD)

13. lanyard or key ring? Keyring wtf I’m not working in a lab haha

14. favorite non-chocolate candy? Cherry flavored lollipops

15. favorite book you read as a school assignment? La Petite Fille de Monsieur Lihn (I think that the title is correct)

16. most comfortable position to sit in? Sit with legs up

17. most frequently worn pair of shoes? I have very simple taste in shoes, they are Vans-like blue shoes

18. ideal weather? First of all, not above 25°C (≈75°F) because I really hate heat (for the legit reason that is UNCOMFORT) and I really hate scorching sun (for the legit reason that is SUNBURNS) ; Second of all, a little windy but not too much, yes I’m a whiny person, also some humidity is great, ANYWAY,,,

19. sleeping position? On my back or on the left side

20. preferred place to write (i.e., in a note book, on your laptop, sketchpad, post-it notes, etc.)? I only type on my laptop or phone, even if I’d love to have the habit of actually writing down. My preferred spots then are on my desk in my bedroom if I write on my laptop, or on the train if I write on my phone (most of my Frozen prompts are written during my 2 hours long commute!)

21. obsession from childhood? I was scared af of the grids at the bottom of swimming pools and absolutely refused to swim at less than 3 meters from them

22. role model? Do fictional characters count…?

23. strange habits? I go to appointments extremely early (sometimes a full useless hour before) because I’m very ashamed when I’m late and want to avoid that ; there are things that I love to say out loud – some feedback about what I see on a screen for example – just like there are things that I  

24. favorite crystal? Any bright and deep colored one

25. first song you remember hearing? I think it was a song from Adibou hjdskhfdjs good jam

[wzbcpromo]

WZBC Rock Top 30: 05 February 2019

1. Daddy Issues, Deep Dream // 2017 Infinity Cat Recordings

2. illuminati hotties, Kiss Yr Frenemies // 2018 Tiny Engines

3. Alvvays, Alvvays // 2014 Transgressive

4. Dude York, Sincerely // 2017 Hardly Art

5. Vundabar, Gawk, A Perfect Sun // 2015 Gawk Records

6. The Spirit of the Beehive, Hypnic Jerks // 2018 Tiny Engines

7. The Beths, Future Me Hates Me // 2018 Carpark Records

8. Forest Hills, Freddy Has a Tuba // 2018 Dark Star Recording, Inc.

9. The Twilight Sad, It Won/T Be Like This All the Time // 2019 Rock Action Records

10. Horse Jumper of Love, Horse Jumper of Love // 2017 Joy Void

11. Sobs, Catflap, EP // 2017 Middle Class Cigars

12. Slow Pulp, Ep2, EP // 2017 Slow Pulp

13. Cayetana, Nervous Like Me // 2014 Tiny Engines

14. Shady Lady, Shady Lady EP // 2018 Turtle Island

15. Part Time, Virgo’s Maze // 2015 Burger Records

16. Lady Pills, Despite // 2016 Babe City Records

17. White Fence, I Have to Feed Larry’s Hawk // 2019 Drag City

18. Partner, Saturday the 14th - EP // 2019 You’ve Changed Records

19. Girlpool, What Chaos is Imaginary // 2019 ANTI-

20. Juice, Workin’ on Lovin’ EP // 2018 Juice Records LLC

21. Mustard Service, Zest Pop // 2017 686500 Records DK

22. Lisa Prank, Adult Teen // 2017 Father/Daughter Records

23. Death Valley Girls, Darkness Reigns // 2018 Suicide Squeeze

24. Mini Dresses, Heaven Sent // 2019 Joy Void

25. Geowulf, Great Big Blue // 2018 37 Adventures

26. Greenleaf, Hear the Rivers // 2018 Napalm Records

27. Mint Green, Growth EP // 2016 Mint Green

28. Naked Giants, Green Fuzz // 2019 Naked Giants

29. Ricky Eat Acid, Haunt U Forever // 2011 Ricky Eat Acid

30. Nite Jewel, Next Thing // 2016 Gloriette Records

[surveysonfleek]

1155.

1. What was the last job you applied for? it was for an online company. didn’t get it. 2. When was the last time you stayed home from work or school? For what reason? i faked sick. there was a huge accident on the way to work and i got too lazy to sit in traffic 1.5hr longer than i usually do. 3. Do you ever have reactions that are inappropriate for the situation at hand (ex - laughing during a very serious situation, etc)? probably as a kid. i’m better at controlling my emotions now. 4. What was the last new TV show you started watching and what did you think of it? i started watching this show, electric dreams. it’s a little like black mirror. the first ep was pretty boring. 5. How do you feel about people posting lyrics as Facebook statuses/tweets/etc? i don’t see this that often.

6. Who last owed you money? my sister, who paid it back asap lol. 7. What’s the latest you’ve ever had dinner? like 10pm maybe? 8. Is there something that your friends do unconsciously that you really love (ex - watching them get excited over something they like)? their laughs i guess. 9. Have you ever had a tarot reading or palm reading? nope. 10. What was the last thing you gave up on? myself lol. 11. When was the last time you had a headache? about a week ago. i never get headaches so i complained about it nonstop. 12. What was the last thing you ordered online? glasses. i just got a new prescription and didn’t want to shell out too much money so i bought a cheap pair to see how it goes. 13. Would you rather be too busy or never have anything to do? Why? lately i’ve been too busy so i just want a day where i do absolutely nothing. when i have nothing to do i’d rather be busy. meh lol. 14. Can you describe the last time you felt like time was moving impossibly slow? at work. always. 15. When was the last time you encountered a puppy? it’s been awhile actually! i think i saw one while driving. 16. What is your favorite kind of soda/juice? soda - root beer or vanilla coke. juice - fresh oj with pulp. 17. What is the worst injury you’ve ever gotten? probably fracturing my pinky. 18. Is there anything that happened a long time ago that you still laugh about? always!    19. Do you ever try to interpret your dreams? haha not really. i’ve always wondered but i’ve never gone out of my way to google it. 20. Do you have any mild food allergies? lactose intolerance. 21. What was the last thing you bought impulsively? dry shampoo. why not. 22. How do you feel about singing songs out loud in front of other people? no thanks. 23. When was the last time you were feeling really, really nervous? always. everyday. 24. If you’re no longer in school, what is something you miss about it? If you’re still in school, what’s something you think you’re going to miss about it? i actually don’t miss much about it apart from the fact life was much simpler. 25. What is the greatest amount of money you’ve spent on a concert ticket? How much would you be willing to spend to see your favorite band/artist? $150 i think. but when you account festivals like tomorrowland and coachella i’ve spent a ton more. i’d spend no more than $200. i’ve seen all my favourite artists tbh. 26. Do you use your turn signals when you’re driving? duh. it pisses me off when people don’t. 27. How did you get your last bruise? i forgot. 28. Have you ever heard a recording of your voice? What did you think of it? yes haha. i hate it.    29. Do you have any irrational fears? cockroaches. 30. What’s the longest amount of time you’ve been stuck in traffic? two hours.