Author Topic: Tannins in pea shoots and pods  (Read 183 times)

Klaus Brugger

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Tannins in pea shoots and pods
« on: 2020-12-26, 05:31:32 AM »
Last year, I found a single seed with clear testa and hilum in a packet of 'E.F.B. 33' winter peas. I hope that it's a mutant that will allow me to compare shoots and pods of white-flowered/low-tannin and purple-flowered/high-tannin peas of the same genetic background.
I'm curious about your opinions on or experiences with white- vs. purple-flowered varieties for shoot and pod use.




I've written this little text with some literature references for a different platform, but maybe it's interesting for some of you:

In peas, a lack of pigments in hilum and seed coat is associated with seeds* of lower tannin levels and, consequently, a “sweeter” taste [1]. The same genotype confers a lack of anthocyanins in the whole plant and, presumably, also reduced tannins throughout the plant. For this, however, I only have “anecdotal evidence”, like white-flowered forage peas being marketed as more palatable and purple mangetouts often having a rather astringent taste. Interestingly, in a tasting of pea shoots of one purple-flowered and three white-flowered winter peas, the purple-flowered variety ('E.F.B. 33') came out on top [2]. In this picture, you see both the pigmented seeds of 'E.F.B. 33' and seeds grown from a single seed with clear hilum/seed coat I found in a packet of 'E.F.B. 33'. I hope that this line proves to be a mutant only differing from 'E.F.B. 33' at the locus of interest (comparing UPOV characteristics will at least enable me to make a guess). Having such “near-isogenic lines” available could permit conclusions about taste differences between shoots of low tannin and high tannin peas.
 
Generally, near-isogenic lines are lines possessing almost, but not quite, the same genotype as their sister or parent line. Typically, they are generated by recurrent backcrossing where after several generations they differ from their recurrent parent merely at the locus under selection. They then can be used in phenotyping experiments to determine the effects of polymorphisms at this one locus.

Another reason why it would be nice to have a white-flowered line near-isogenic to 'E.F.B. 33' is because 'E.F.B. 33' ranks among the most winterhardy pea cultivars. Winterhardiness seems to be linked to presence of anthocyanins in traditional winter pea material [3] but this linkage might have been broken in newer varieties. Today, several white-flowered varieties with good winterhardiness exist, some even being semi-leafless, a trait morphologically associated with a higher susceptibility to freezing injury as shown in experiments with – yes! – near-isogenic lines [4].

*While tannins are present in the seed coat, in the cotyledons bitter tasting saponins play a more important role [5].

[1] Clark, S. (2019): Pea (Pisum sativum L.) Characteristics for Use and Successful Planting. USDA, NRCS, Big Flats, NY. Plant Materials Technical Note No. 19-01.
[2] ARCHE NOAH (2019): Aktivitätsbericht Zuckererbse. Sorten- und Produktentwicklungen aus Gemüseraritäten in der Region Kamptal in einem partizipativen Prozess: LEADER-Projekt März 2016 – Februar 2019.
[3] Markarian, D., Harwood, R.R., & Rowe, P.R. (1968): The inheritance of winter hardiness in Pisum II. Description and release of advance generation breeding lines. Euphytica, 17, pp. 110–113.
[4] Étévé, G. (1985): Breeding for Cold Tolerance and Winter Hardiness in Pea. In: P.D. Hebblethwaite, M.C. Heath, & T.C. Dawkins (Eds.): The Pea Crop: A Basis for Improvement London, Butterworths, pp. 131–136.
[5] Tulbek, M.C., Lam, R.S.H., Wang, Y.(C.), Asavajaru, P., & Lam, A. (2017): Pea: A Sustainable Vegetable Protein Crop. In: S.R. Nadathur, J.P.D. Wanasundara and L. Scanlin (Eds.): Sustainable Protein Sources, London, Academic Press, pp. 145–164. 

Andrew Barney

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Re: Tannins in pea shoots and pods
« Reply #1 on: 2020-12-26, 09:43:23 AM »
Interesting thread! While a lot of people seem to think vegetable varieties with anthocyanins can taste bitter I have found that while some do, most do not, and therefore I suspect it is not the anthocyanins themselves that might taste bitter, but instead genetic drag that might be only slightly linked from an unimproved wild-type background. I think this bitterness (if any) linkage can be broken very easily.

More often than not I find fruits and vegetables with anthocyanins to taste better, not worse. But it is very interesting. I suspect the trend for white flowers and seeds and vegetables lacking color might just be an unintended side effect of inbreeding. There was a Siberian experiment to domesticate foxes like dogs and the result was that surprisingly in very few generations they became tame and started showing domesticated traits like white fur, floppy ears, softer eyes, curly tails, and more infant like appearance. I suspect something similar can happen in plants.
« Last Edit: 2020-12-26, 09:46:18 AM by Andrew Barney »

Andrew Barney

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Re: Tannins in pea shoots and pods
« Reply #2 on: 2020-12-26, 09:51:04 AM »
I haven't used peas for shoots that often,  though maybe I should. Since most purple podded peas do not taste good themselves I suspect they would not taste good as shoots. They would at least need to have low fiber genes in the pod which I think also affects the tendrils.

The only two purple podded varieties I like are Sugar Magnolia and Midnight Snow (which I'm using for my red podded pea improvement line).

I have heard deer prefer white flowered snap peas over purple flowered field peas. But again that might be due to genetic drag of an unimproved wild-type, not necessarily the flower colors themselves.
« Last Edit: 2020-12-26, 09:55:05 AM by Andrew Barney »

Steph S

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Re: Tannins in pea shoots and pods
« Reply #3 on: 2020-12-30, 02:06:08 PM »
Klaus, that is a great project and what luck to have found a near isogenic line to play with.   Let us know what you think of the taste of the sprouts.
I have heard it said that peas with dark seeds (from anthocyanins presumably but perhaps tannins as well?) are hardier - more tolerant of cold soils etc.
This does make sense, anthocyanins are one of the plant secondary substances which they are able to use to raise the freezing point in their tissues.

Do you have a simple test (besides taste) to evaluate the level of tannins in the two plants?

Andrew Barney

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Re: Tannins in pea shoots and pods
« Reply #4 on: 2021-01-02, 01:33:19 PM »

Do you have a simple test (besides taste) to evaluate the level of tannins in the two plants?

I wonder if a color chromatography test could work? With alcohol or acetone.

Steph S

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Re: Tannins in pea shoots and pods
« Reply #5 on: 2021-01-02, 02:40:11 PM »
I wonder if a color chromatography test could work? With alcohol or acetone.
I seem to remember doing chlorophyll with acetone in the lab but that was a long time ago.  Maybe it would separate them?  Just googled 'paper chromatography tannins' and there seems to be lots written about that - good call.  I guess chlorophyll and anthocyanin etc would be well separated from any tannins that way.

There are a few other tests described (a bit vaguely) at the tannins wiki.    A higher concentration of tannin would make a blacker/darker "ink" reaction with ferrous sulphate.   Described as 'Goldbeater's test' using oxhide, but the same test can be done with paper or cotton - may need a longer and/or hot soak in a good strong solution of the pea shoots.   I guess chromatography could be used to evaluate the results, or just a qualitative judgment should be possible if that's all required.
https://en.wikipedia.org/wiki/Tannin
The reaction with FeS is a specific test for tannins, I would probably try that just because I have some on hand (vs acetone, do not have!)  ;)


Klaus Brugger

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Re: Tannins in pea shoots and pods
« Reply #6 on: 2021-01-10, 11:36:35 AM »
Thank you for your interesting answers, very much appreciated!
It would be really cool to have an easy test for tannins giving reliable results. I've only looked into it very quickly and found suitability for condensed tannins, specificity (specific for phenolic compounds not being helpful when tannins are associated with anthocyanins ...), and hazards of used substances when not working in an appropriate lab to be obstacles. I will look into your suggestions, thank you very much! :-)

Steph S

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Re: Tannins in pea shoots and pods
« Reply #7 on: 2021-01-10, 03:56:52 PM »
Separating the effect of anthocyanins may not be a problem if you extract your tannins in hot water.  But that depends on the stability and concentration of the anthocyanins - you can easily test it by boiling the colored parts and determine if the antho color fades easily or persists as a color in the water.

I confess my interest in tannins comes from barking (and dyeing) a bunch of stuff this year, so I've been reading about them all fall.  Anthocyanins can make beautiful prints or dyes but are easily destroyed by heat in most cases, or by exposure to air, light etc.  They also do react with iron to make violet blue or grey colors.  Concentration woud be the key, anything deeply colored and concentrated is more likely to persist and so extracting the tannins in hot solution would not destroy them.   We got great extracts from bark in an alkaline solution (1/2 tsp of soda ash in a 1.5- 2 liter crockpot at around 180 F for an hour).  I also read that baking soda was added to the bark pot when extracting for barking nets.  But you may likely still get the full tannin extract from your shoots in plain water - cold water takes a good bit longer if you go that route.  Just pouring boiling water over the shoots and let stand until cool or overnight would be adequate I would think, since it's not woody material full of lignins etc.  Hot water also denatures proteins so that would tend to release any tannin complexed with protein.

Here is an example of a process to compare different tannins with iron:
https://www.suzannedekel.com/post/tannins-and-ferrous-sulfate-research
All of those plant tannins are readily available to use as a standard for comparison, or you can also get 'pure' tannin from chemical supply houses, then make up your samples by dry weight for direct comparison of the intensity of color.

OTOH complexing the tannin with gelatin to make a precipitate could be an even better method for your purpose.
Some references:
https://pubs.acs.org/doi/pdf/10.1021/jf60157a017
gives a simple method - I would extract some weight of the plant material in hot water, then cool it, mix with gelatin powder and allow to stand for an hour or two - I guess the challenge would be to decant and dry the precipitate in order to weigh it?   Or the tannin (now separated from anthocyanins etc) could then be removed from the gelatin (probably by heating? ) to get a tannin solution which can be evaluated by another means, such as the reaction with iron. 

If you want to invest in a sensitive method, this radial diffusion method requires agar plates containing protein which I guess you'd have to buy, but it sounds really easy to quantify the plant tannins - you put the extract in a well in the middle and then measure the diameter of the ring that forms.  And it says anthocyanins and such do not affect the results.  Could be the simplest approach.
https://link.springer.com/article/10.1007/BF01880091


Andrew Barney

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Re: Tannins in pea shoots and pods
« Reply #8 on: 2021-01-10, 04:54:00 PM »
I've extracted concentrated purple anthocyanins from boiling corn husks from my deeply purple Indian corn population in the past. I only boiled it as long as it took for the water to be saturated so I have no idea if the anthocyanins would have broken down with more heat boiled for a long time. I left it in the refrigerator for several weeks and found it was starting to ferment (which i found to be very fascinating). I wonder sometimes if those anthocyanins would have turned into actual ethanol had i left the process to continue or whether that liquid would have been a good sourdough starter for wild yeast.

I would have loved to use the water to dye something in, but I do not know what the best mordant to use would be so that the color would be fixed permanently. Any suggestions on the topic would be very welcome. I have morre purple corn husks in the basement just waiting for the same experiment to be done again.

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Re: Tannins in pea shoots and pods
« Reply #9 on: 2021-01-10, 06:36:24 PM »
Those are great looking corn husks, Andrew!   :D 
Potassium alum is the best mordant to use, you will easily find sources if you search for natural dye.  Relatively non toxic and it works.  Usually it's recommended for wool and silk (protein fibers) while alum acetate is preferred for cotton unless you do a multistep process with tannin and potassium alum, or quite a long process with repeated soaks in soy milk or similar.  Potassium alum can work with cotton but most important to soak it in the mordant for more than 2 days.  Otherwise you will only get pale colors.
The amount of alum to use is based on weight of fiber, and the amount of plant material is also reckoned on wof - dry weight for both, you may well find recipes out there for corn husks as well (I haven't used myself since I've never grown any).  It takes about 4 tsp (approx 15 g) of potassium alum for 100 g dry weight of fiber.
If you want intense color I do recommend contact print or 'ecoprint' as a method, or 'bundle dyeing'.  It takes a lot of plant material even to get a pastel shade with anthocyanins used as an extracted dye, and they also tend to fade pretty quickly.  Extracting as you did or just pouring boiling water and letting it steep like a tea is best.  Some nice solar dyes are possible I think (although IDK how well they hold up to light and washing) which avoids the higher heat in the dye pot.   Just put your mordanted fabric or plant/fabric bundle into the dye jar such as you have, and leave it in the sun for as long as it takes (weeks likely).
I've been happily surprised with some contact prints involving antho - dried hollyhock petals produced some really intense purples and blues after a quick moistening with vinegar, and seems washfast enough.  The plant material is applied directly to the wet, mordanted cloth, and rolled up tight to provide some pressure, then steamed for an hour or two.  I think some less soluble things in the plant material as well as the tannins also come out through the long contact process and help to fix the dyestuffs.   
In general, plant material that is high in tannins prints well, while dyestuffs alone will just flow and make blobs of color.
I haven't tried to print pea shoots but that was my first thought - see which one prints better.  ;)

Klaus Brugger

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Re: Tannins in pea shoots and pods
« Reply #10 on: 2021-01-11, 03:39:02 AM »
...
If you want to invest in a sensitive method, this radial diffusion method requires agar plates containing protein which I guess you'd have to buy, but it sounds really easy to quantify the plant tannins - you put the extract in a well in the middle and then measure the diameter of the ring that forms.  And it says anthocyanins and such do not affect the results.  Could be the simplest approach.
https://link.springer.com/article/10.1007/BF01880091

Thanks again for all this great information! I really like the assay method described in the last paper. It would be much easier having just standard lab equipment (I don't even have piston-driven pipettes at hand), but I guess it should be possible even "at home". I just saw that there are also really cool ready-to-use microplate assay kits for vegetable proteins that probably wouldn't even be more expensive. But for these I'd need at least a microplate reader and probably a centrifuge. There's an open "group lab" (used for "DYI biology" etc.) a 2.5 hour drive away from me. I'll have to check which equipment they have.