# Grass sap, WSC, and refractometry

Hi all

I am an electronic engineer and am currently looking into taking brix measurements of pastures in NZ. (Not really my speciality!!!)

I am using a brix refractometer which is meant to measure the surcose/water ratio for a solution. Currently I use a garlic press to sqeeze the sap from a grass sample onto the refractometer. I am generally focusing on perennial ryegrass.

1. What are all the compounds that would be squeezed from the grass??? My assumption so far is that it would be water, surcose, fructans, pectins, minerals, protiens, and a small amount of structral carbohydrates ( cell walls etc). Is this correct or are there other major components i'm missing??? What sort of proprotions could I expect these in???

2. If I roll the sample prior to squeezing it I obtain higher brix readings, proportional to how long I roll it for. By rolling/crushing the sample I assume I am introducing more broken up strutural carbohydrates to the solution. Is this correct???

Thanks alot, I appericate any ideas, thoughts related to this.

Cheers
Toby

Ouabache
Homework Helper
Hi all

I am an electronic engineer and am currently looking into taking brix measurements of pastures in NZ. (Not really my speciality!!!)
that okay. it is never too late to learn...

1. What are all the compounds that would be squeezed from the grass??? My assumption so far is that it would be water, surcose, fructans, pectins, minerals, protiens, and a small amount of structral carbohydrates ( cell walls etc). Is this correct or are there other major components i'm missing??? What sort of proprotions could I expect these in???
You have the right list of compounds expected from squeezing liquid from cut grass leaves. I don't know, off hand, the proportions. I do believe the water soluble carbohydrates are a large percentage of your extract. You may want to consult some texts on plant physiology and biochemistry to get a better assessment.

2. If I roll the sample prior to squeezing it I obtain higher brix readings, proportional to how long I roll it for. By rolling/crushing the sample I assume I am introducing more broken up strutural carbohydrates to the solution. Is this correct???

Cheers
Toby
Yes, you are breaking up more cells and allowing their contents to add to the liquid phase already available along the cut surfaces of the grass leaves.

jim mcnamara
Mentor
The expected soluble carbohydrates from ryegrass:
http://cat.inist.fr/?aModele=afficheN&cpsidt=4466646

Time of day has a huge effect on free simple carbohydrate levels in leaves.

You probably want to lyse the leaves fully with a blender, a garlic press is not going to get all of the available simple carbohydrates out of cells and into solution.

Organic acids and plant pigments can distort refractive brix measurements.
Wine makers use brix corrections. Or other methods, fro example.

Maybe if you told us what you are trying to do, other than mash up grass leaves, we could help. :)

The expected soluble carbohydrates from ryegrass:
http://cat.inist.fr/?aModele=afficheN&cpsidt=4466646

Time of day has a huge effect on free simple carbohydrate levels in leaves.

You probably want to lyse the leaves fully with a blender, a garlic press is not going to get all of the available simple carbohydrates out of cells and into solution.

Organic acids and plant pigments can distort refractive brix measurements.
Wine makers use brix corrections. Or other methods, fro example.

Maybe if you told us what you are trying to do, other than mash up grass leaves, we could help. :)

Hey Jim

I am currently looking into using a brix meter to assess the soluble sugars in a pasture. Yes, there are many things which affect the brix reading, time of day, year, barometeric pressure, soil history, you name it...

With regards to blending the grass, I would have thought this would dramatically change the refractive index of the sap and therefore the brix reading? Do you mean blend then filter the grass??

I say this because I am worried by rolling/crushing/cutting the sample, I am introduing more strutural carbohydrates to the solution.

Thanks for your help, What do you think?

jim mcnamara
Mentor
The best and fastest ploy to remove particulates is to centrifuge the junk to the bottom.
Since you are interested only in simple sugars, this moves almost all particulates out of the way in a very short time.

Structural carbos are polymers and are particulates: amylose, cellulose, and lignans are not water soluble. You will find a higher sucrose reading if you frap the grass in a blender vs simply trying to squeeze it out.

Your biggest worry is enzymatic changes, not structural carbohyrates. The longer you let the "squeezate" sit the more amylose will be synthesized from sucrose, for example. Your best choice is to take fresh clippings into the lab, frap them, centrifuge the sludge, then get a brix measurement in the shortest reasonable amount of time. Keep the time between frappage -> brix as constant as possible.

Where are you? Lolium is not a happy camper this time of year in most parts of North America. Lolium also perferentially translocates simple carbos down to the rhizomes where they are stored as starch - during short day periods of the year.

The best and fastest ploy to remove particulates is to centrifuge the junk to the bottom.
Since you are interested only in simple sugars, this moves almost all particulates out of the way in a very short time.

Structural carbos are polymers and are particulates: amylose, cellulose, and lignans are not water soluble. You will find a higher sucrose reading if you frap the grass in a blender vs simply trying to squeeze it out.

Your biggest worry is enzymatic changes, not structural carbohyrates. The longer you let the "squeezate" sit the more amylose will be synthesized from sucrose, for example. Your best choice is to take fresh clippings into the lab, frap them, centrifuge the sludge, then get a brix measurement in the shortest reasonable amount of time. Keep the time between frappage -> brix as constant as possible.

Where are you? Lolium is not a happy camper this time of year in most parts of North America. Lolium also perferentially translocates simple carbos down to the rhizomes where they are stored as starch - during short day periods of the year.

Hey Jim

I am in NZ. It is spring, nearing summer here.

Very interesting, I did not know about enzymatic changes. Does this mean the brix reading will change over time? What exactly is happening in this process?? I guess surcose is being changed to a larger compound or something?

I will definately try the blender/centrifuge technique now. First I have to find a centrifuge. What exactly do you mean by frap them? Do you think after this process the brix reading will be quite close to the actual water soluble sugar content of the grass?

Wouldn't other compounds such as pectin, glucans etc have a large effect on the reading, or would this be centrifuged away as well?

Toby

Ouabache
Homework Helper
What exactly do you mean by frap them?
I suspect he means to whizz it up, in a blender, Frappe is a term for a thick ice cream milkshake, in some parts of the U.S.

I believe as long as you are consistent in how you process your leaf samples, you will obtain valid BRIX readings. You may want to consult the literature and find out if there is a standard procedure. By using that method, it will be easier to compare to others who have also taken similar measurements in pasture grasses.

I believe as long as you are consistent in how you process your leaf samples, you will obtain valid BRIX readings. You may want to consult the literature and find out if there is a standard procedure. By using that method, it will be easier to compare to others who have also taken similar measurements in pasture grasses.

Hey

Ahh so thats what frap means, thanks for that.

I'm not to sure that is correct, that I will obtain a vaild brix reading even with a consistent method. Even though it is consistent with prior readings and maybe other peoples reading (assuming there using the same technique) brix meters have not yet been proven to give an accurate correlation with actual soluble sugar content. This is what I am currently trying to prove. We did initial testing with high and low nitrogen input paddocks, hoping they would be different in soluble content, but the results compared with lab testing were not to flash.

I am sure there is no standard procedure for sampling pasture at this point in time. People everywhere comment they can obtain brix upto 12 to 15 %, but this could simply be a function of there preparation method, ie. rolling a grass sample for >40sec gives a brix of ~12% while not rolling it gives ~4%.

I guess using a centrifuge/blender method will show a correlation, and i hope i can get one somewhere, but the problem is creating a method which farmers can use quickly, easily and accurately.

I duno, I'm an electronic engineer what do I know!

jim mcnamara
Mentor
Suggestion: Consider already massively tested technology

Blood glucose meter - accurate readings, locally available supplies from the pharmacy.

In Canada, the standard unit of blood glucose measure is millimoles per litre (mmol/L). In other countries, it may be milligrams per decilitre (mg/dL), which is 18 times higher than the same results expressed in mmol/L. Some glucose monitors used in Canada display their results only in mmol/L. Other meter models are capable of displaying their results in either unit of measure.

So, ask what setting NZ has for these meters, if there is a standard one.

1. use the garlic press to extract sap.

2. Have a 20* ml plastic vial filled with .1% sucrase (Carolina Biological
supply has it for example) solution.

3. place 1 drop of sap into the solution. Wait 30 seconds, then put one drop
of the solution onto the glucose meter test tab.

4. the mmol/L reading appears in circa 5 seconds and is more accurate than refractometry.

* you will have to experiment with this volume to get readings in the range
the meter will interpret: 20 - 500 mmol/L (or whatever). If you work those levels out correctly it can translate directly to a percent the farmer will intuitively understand.
Plus, splitting sucrose into 2 glucose molecules increases the concentration of sugar so it becomes easier to get a usable dilution factor.

Supplies the farmer in the field needs
Glucose meter
Glucose test tabs
Premixed vials of enzyme - farmer can mix this in the kitchen sink.
garlic press

Back in the shed the farmer needs:
Small bottle of standard glucose mixture for testing dilution factor.

This will work reliably unless you are researching this for a refractometer company :)

Yes, smart idea, I had researched into other alternatives

However I thought a blood meter measured specifically glucose though some sort of chemical reaction.

Sucrase would seperate surcose, but would it seperate fructans???
In this case, glucose is only a small portion of the total sugars in a grass sap sample. I found literature which stated glucose and fructose make up a small portion, surcose makes up a slighlty larger portion, and fructans make up a very large portion of the total sugars in grass. I haven't found anything else to back this up but it seems reasonable. Seperating all this out would mean quite a large amount of fructose compared with glucose???

So would a blood meter would not work in this case, as a sqeezed sample not majorly contain glucose???

Last edited:
Do you have an update on how you got on trying to measure the amount of sugars in grass? I am trying to do something similar, to see how much variation there is in total sugar content through a day. I would like a fairly easy method to show how the relative amount changes with time of day and have been experimenting with a refractometer, not much luck so far as my reading stayed fairly constant throughout the day.

jim mcnamara
Mentor
see:
Code:
Title: Water soluble carbohydrate content of grass.
Personal Authors: Appleton, M.
Author Affiliation: Liscombe Exp. Husbandry Farm, Dulverton, Somerset TA22 9P2, UK.
Editors: No editors
Document Title: Sixth silage conference 1981.

Abstract:
Diurnal changes in water-sol. carbohydrate content (WSC) in a perennial
ryegrass cv. S24/white clover sward were monitored at 0900, 1300 and 1700 h
and on several occasions during the night from 17 May-15 June 1980.
WSC was highest at 1700 h and this was correlated with max. DM content.
It was concluded that there was no opt. cutting time with regard to sugar content
but that DM content at time of cutting is a more important factor.

Publisher: Edinburgh School of Agriculture.