A Comparative Trial of the Pollen Subs: Half 5- Revisiting de Groot

 Contents

Diving into de groot

Understanding what De Groot did

The issue with normalizing to tryptophan

Re-interpreting de groot’s findings

Protein conversion — Patty into bees

Justus von Liebig’s Legislation of the Minimal and important amino acids

My eating regimen calculator

So how in regards to the examined diets?

The homebrew formulation

Not but executed …

References

A COMPARATIVE TRIAL OF THE POLLEN SUBS

Half 5: Revisiting de Groot

Beekeeper-Funded Analysis

Randy Oliver^a and Vincent Ricigliano^b
aScientificBeekeeping.com, ^bUSDA ARS

First printed in ABJ November 2021

Any apicultural paper that mentions the amino acid necessities of honey bees references the monumental landmark examine by Antonius de Groot printed in 1953. However that was early in our scientific understanding of animal vitamin and synthetic diets. I’ve nothing however admiration of de Groot’s work, particularly since he was fairly clear in regards to the limitations, shortcomings, and interpretation of his pioneering examine. Nonetheless, I really feel that it’s maybe time to reinterpret his findings.

DIVING INTO DE GROOT

De Groot was an excellent, meticulous, and devoted researcher, whose monumental work [[1]] is astounding and spectacular. I’ve no criticism in any way of any a part of his 80-page paper, as a result of as you’ll see, he was very cautious about drawing onerous conclusions from what he understood to be pioneering analysis. The primary 5 chapters of his paper cowl the state of information at the moment about honey bee and bug vitamin, by which he states that “the information concerning the amino acid necessities of bugs remains to be solely fragmentary.”

He reviewed earlier analysis indicating that expeller-processed soybean flour, brewer’s yeast, cow’s milk, and rooster egg have been helpful elements in synthetic diets for honey bees. He spent an excessive amount of time evaluating varied experimental strategies that could possibly be used to find out the dietary adequacy of a eating regimen, akin to the event of the hypopharyngeal glands or longevity of grownup caged bees. Nonetheless, as he explains, “testing for longevity doesn’t inform us the vitamin that bees require to develop from egg to grownup.” Ideally, one would examine the dietary necessities for rising larvae by means of to pupation, however sadly, nobody had but discovered a way for rising them on synthetic diets.

So de Groot settled upon “learning the expansion of simply emerged bees on completely different diets with assistance from weight and nitrogen determinations,” following the findings of Haydak in 1934 (Determine 1).

Fig. 1 Newly-emerged employee bees free in a colony start consuming some pollen shortly after emergence, after which eat ravenously after round 40 hours [[2]]. Throughout this time they add proteinaceous tissue to their our bodies (as measured by nitrogen content material) for just a few extra days, gaining roughly an extra third of nitrogen content material over that at emergence. Knowledge plotted from Haydak [[3]].

The above weight good points are largely as a result of growth of the hypopharyngeal (brood meals) glands within the head, the fats our bodies within the stomach, and eventually the event of the wing muscle groups within the thorax.

Sensible software: This prodigious price of tissue progress within the emerged grownup employee requires good conversion of the protein from a highly-nutritious eating regimen.

De Groot’s preliminary analysis indicated {that a} mixture of casein (milk protein) and sucrose alone provided an ample eating regimen for employee post-emergence growth. That is slightly stunning since he didn’t discover a want for lipids, sterols, nutritional vitamins or minerals in his experimental diets, presumably as a result of the employees emerged with sufficient physique reserves of these vitamins for additional growth on a eating regimen consisting solely of mixtures of pure amino acids (replicating these in casein) and sucrose.

Sensible software: This raises the query of whether or not testing diets on newly-emerged staff is definitely the very best take a look at of a man-made eating regimen, since these staff already comprise reserves of important vitamins that they will get by with out for a while. I’ll later return to this topic of how we must be testing diets designed to promote colony progress as a complete, slightly than solely grownup bee weight achieve.

It isn’t till web page 65 that de Groot begins the exams to find out the “Quantitative Amino Acid Necessities” ratios for the expansion of newly-emerged staff. With that objective in thoughts, de Groot measured the adjustments in dry weight and nitrogen content material (as proxy for protein content material) of caged newly-emerged employee bees then fed managed diets. Though they grew greatest on a eating regimen of pure pollen, they grew almost as properly on diets of “vitamin-free” casein (milk protein). Surprisingly, the organic worth (the % of nitrogen within the eating regimen that was retained within the bees’ physique tissue) was higher for the substitute diets than for pure pollen.

Sensible software: Though de Groot’s findings have been for caged bees and an unnamed pollen, the bees wanted to eat way more protein within the type of pollen to acquire the identical quantity of progress as when the protein or amino acids have been offered in pure type. So though pollen is the pure supply of vitamin for bees, the protein in a man-made eating regimen could also be extra effectively digested and/or utilized.

Then de Groot shifted to utilizing acid-hydrolyzed casein (milk protein) as an amino acid supply, and since tryptophan is destroyed throughout hydrolysis, found that it was an indispensable amino acid. From this level on, he fed his take a look at bees diets of sucrose and mixtures of amino acids, so as to decide which amino acids, if missing, would stop progress, after which, in a sequence of meticulous experiments, the minimal quantity of that EAA that wanted to be added again to permit for “regular” progress.

UNDERSTANDING WHAT DE GROOT DID

De Groot was now prepared to maneuver on to figuring out precisely how a lot of every particular person EAA was required in a eating regimen. He painstakingly measured the will increase in dry weight and physique protein of samples of caged newly-emerged employee bees fed standardized synthetic diets of amino acids (based mostly upon casein), every formulated with as much as 9 completely different concentrations of the one amino acid to be examined. To do that, after feeding, he tediously dissected out the alimentary observe from the bees in every pattern so as to take away any intestine contents, dried the remaining bee carcasses, weighed them, after which ran chemical nitrogen analyses. And he did this for at least 8 replicates of 5 bees per take a look at, for all 10 EAAs — almost 400 tedious exams and analyses in all.

I graphed his information for isoleucine for example how he interpreted his outcomes (Determine 2).

Fig. 2 This graph exhibits the rise in bee physique nitrogen from feeding a eating regimen of sugar supplemented with a combination of 18 amino acids at 3% of the load of the sugar, with the proportion of isoleucine (relative to the full quantity of amino acids) starting from zero to eight%. Notice the inflection level within the curve at 4% on the x-axis, indicating that there was no profit to rising the proportion of isoleucine to above 4% of the full amino acid combination.

De Groot then plotted graphs for all 10 EAAs (Determine 3):

Fig. 3 The principal and most-cited discovering from de Groot’s analysis come from the above chart, with the y-axis indicating nitrogen content material, and the x-axis displaying the proportion of that amino acid relative to the full. The 2 strains point out feeding at both of two amino acid-to-sugar proportions — 2% (dotted) or 3% (strong). De Groot defined, “Every curve exhibits an increase with rising focus till an extra enhance is now not accompanied by an upward development of the curve. Within the latter area the focus is above its minimal stage for maximal progress. Clearly, this minimal stage is located close to the angle of the graphs.” Supply [[1]].

Sensible software: The explanation that I included the determine above is that these curves are the premise for de Groot’s widely-cited amino acid proportions, the purpose being that these “onerous” numbers have been based mostly upon his eyeball interpretation of slightly crude information plots.

However these minimal ranges additionally got here with an vital disclaimer. Enable me to once more quote de Groot immediately:

It could be emphasised, nevertheless, that the noticed minimal concentrations for maximal progress don’t have anything to do with the minimal required consumption for maximal progress underneath pure circumstances. They solely characterize the minimal quantities which, together with the quantities of the opposite amino acids, induce a progress price maximal underneath the circumstances of those experiments. Despite the fact that we aren’t justified in attaching absolute worth to those minimal concentrations, they’re invaluable together with one another. From the minimal concentrations supporting maximal progress, an approximation of a proportionality sample of important amino acids will be deduced, supplying data concerning the relative portions that are required for protein synthesis [boldface mine].

Sensible software: De Groot was very clear that his findings shouldn’t be taken as gospel, however slightly as recommended steerage. And he makes very clear that the oft-cited “minimal stage required in honeybee” in his Desk 25 [[4]], will not be for figuring out the optimum proportion in a formulated eating regimen, however slightly to concentrate to the proportions of the EAAs relative to one another.

However one should remember that in his research, these ranges have been estimated from very tough weight achieve and physique nitrogen content material curves of grownup bees fed a 2 or 3% (relative to sugar) extremely synthetic combination of 18 important and nonessential amino acids “approximating the composition of casein.” However what is usually missed is that de Groot himself was clear that bee progress was suboptimal on this eating regimen:

Nonetheless, it could be famous, that because of the focus of the nitrogen compounds within the diets employed, progress was suboptimal. Whether or not the identical applies to circumstances enabling a better progress price stays to be investigated. Subsequently, we’re justified in concluding solely, that underneath the circumstances involved, a ration containing 18 amino acids supported a progress price equal to that obtained with a comparable quantity of casein.

And actually, bees grew barely higher on an “isonitrogenous” eating regimen of pure pollen. De Groot’s statement that grownup bee progress was suboptimal on his experimental diets have to be saved in thoughts. The protein-to-carbohydrate ratio on the highest examined focus was 3% (1:33). Altaye [[5]], permitting bees to steadiness their very own protein-to-carbohydrate ratio discovered that bees focused a 1:12 ratio when the offered protein supply was casein. De Groot’s take a look at diets have been clearly protein restricted.

De Groot additionally made clear that:

Beneath the circumstances of the above described experiments, the speed of progress is comparatively sluggish as in contrast with that noticed underneath pure circumstances.

He explains that he used the low concentrations of amino acids relative to sugar so as to keep away from the poisonous results of protein overload noticed in longevity experiments. He additionally notes that among the non-essential amino acids might exert stimulatory results underneath circumstances of “regular” progress — notably serine, glycine, and proline.

Sensible software: It’s extra vital to concentrate to de Groot’s proportionality relationships for the EAAs slightly than to the absolute values for every EAAs proportion of the full protein as listed in his Desk 25.

THE PROBLEM WITH NORMALIZING TO TRYPTOPHAN

Early in his analysis, de Groot saved having tryptophan come on his radar, primarily as a result of it’s destroyed by acid hydrolysis of a protein into its constituent amino acids, and thus wanted to be added again in experimental diets. Tryptophan is talked about 75 occasions in his examine, and by point out 47 he’d already put it on the high of his record of EAAs. So when tryptophan occurred to come back in on the 1% stage, de Groot used that worth as “unity” to check all the opposite EAA minimal ranges to.

Sensible software: That arbitrary resolution by de Groot to make use of tryptophan because the reference EAA was unlucky as a result of the quantification of tryptophan is harder than for the opposite amino acids (since it’s destroyed throughout acid hydrolysis). Subsequently, many printed amino acid profiles for pure pollens [[6]] and feedstuffs lack a worth for tryptophan. However much more importantly, tryptophan will not be the main EAA required in bee diets.

We’ve realized a bit about animal vitamin since 1953. So let’s again up a bit.

RE-INTERPRETING DE GROOT’S FINDINGS

Is there a greater EAA to make use of for reference as an alternative of tryptophan? I recommend that we again up 15 pages in his paper. In his preliminary analysis, De Groot had ready 17 diets, every poor in a single amino acid, to see how properly newly-emerged bees gained weight (Determine 4) or elevated nitrogen content material (not proven).

Fig 4 This graph of bee weight in response to amino acid deficiencies is how de Groot decided which amino acids have been “important.” Notice how bees grew “usually” within the higher group, on diets missing any of the non-essential amino acids (“full” contained all 17), however ceased gaining weight within the decrease group, as a consequence of these diets missing one of many ten “indispensable” or “important” amino acids. Supply [[1]].

Sensible software: The important thing take-home from the graph and knowledge above is that bees can develop fairly properly for the primary week of their lives even with a deficiency of 1 EAA, however within the second week a deficiency in essentially the most important amino acids turns into obvious. Notice the important significance of valine, threonine, and leucine (noticing this was my aha second). Since leucine is the EAA required in biggest proportion in a honey bee eating regimen (and the EAA in highest proportion in all of the pollen subs that I examined apart from Homebrew), it occurred to me that maybe we must always use leucine, as an alternative of tryptophan, because the reference EAA when analyzing diets for EAA steadiness (Determine 5).

Fig. 5 Leucine is the predominant EAA required for honey bee progress, and thus determines the proportion of whole protein that will get utilized. Since it is usually the predominant EAA in most feedstuffs utilized in pollen subs, I suggest that it must be the EAA to which we examine the quantities of the opposite EAAs so as to decide EAA deficiencies.

De Groot’s analysis recommended that leucine ought to represent 16% of whole EAAs. Of curiosity is that of the diets that I examined, the highest performers have been those who have been closest to that proportion (Determine 6).

Fig. 6 Is it simply by probability that the efficiency rating of the examined subs nearly precisely adopted how shut their leucine proportion was to de Groot’s advice? Did the poorer-performing diets contained an extraof leucine, or have been they as an alternative poor within the different EAAs?

Sensible software: Refer again to Determine 4 to see how vital the predominant EAA, leucine, was for bee weight achieve. So maybe what we must be focusing upon is a eating regimen’s means at changing leucine into bee physique mass.

PROTEIN CONVERSION — PATTY INTO BEES

Nutritionists typically calculate a eating regimen’s Protein Effectivity Ratio (PER) — the achieve in physique mass of the fed animal, divided by the quantity of protein within the take a look at eating regimen. I didn’t weigh the bee cluster in each hive, nor did most colonies develop in power throughout the first half of the trial, however based mostly upon the quantity of patties consumed, their protein contents, and the change in cluster dimension, I might very roughly calculate approximate figures for the effectivity in protein conversion for every take a look at eating regimen to cluster dimension (Determine 7).

Fig. 7 Please take the outcomes above with a giant grain of salt, as a result of variety of variables concerned. However because the error bars for the 18 hives in every take a look at group are actually small, the info recommend that the protein within the merchandise to the left (which occurred to be the best-performing diets) was utilized extra effectively than in these to the appropriate, at the least throughout the late-summer feeding interval.

Sensible query: Why was the protein in some diets extra effectively transformed into bees than in others? Evaluate the above graph to Determine 6, and observe how diets with extra leucine have been poorly utilized. This brings us to the topic of:

JUSTUS VON LIEBIG’S LAW OF THE MINIMUM AND ESSENTIAL AMINO ACIDS

Within the late 1800s, the above-named agricultural chemist proposed a groundbreaking idea concerning the fertilization of crops. He identified that essentially the most environment friendly use of vitamins was to not dump on an extra of anyone, however slightly that the quantity of progress is decided by the important nutrient in shortest provide, whichever nutrient it could be.

Then within the Thirties, W.C. Rose printed research by which they discovered that it was potential to determine sure amino acids as being “important” for animal progress [[7]]. Simply as von Liebig had realized that optimum plant progress will be attained by addressing any “limiting vitamins,” this idea additionally utilized to the expansion of animals, and in our case, to the expansion of honey bees and their colonies (Determine 8).

Fig. 8 Within the case of honey bee diets and the ten EAAs, think about the barrel above as having solely ten staves, and {that a} eating regimen by which the EAAs have been in excellent steadiness would fill to the brim. A very powerful stave would doubtless characterize the predominant EAA, leucine, with the opposite stave heights representing the proportion of one another EAA if it have been within the appropriate ratio to completely complement leucine [[8]]. In diets with an extra of a sure EAA, that stave can be above the utmost fill stage (decided by the leucine stave), whereas EAAs in deficit can be beneath the leucine brim, and never permit full utilization of the leucine.

Sensible software: It’s all about offering the optimum “steadiness” of vitamins, slightly than an underutilized extra of anyone.

For me to visualise EAA deficiencies, I created a calculator with considerably related “staves.” So for instance, suppose you made a patty of equal components of soy flour and brewer’s yeast (plus sugar after all), the EAA ratios would seem like the “staves” beneath (Determine 9).

Fig. 9 The EAA proportions of a soy/yeast eating regimen. The grey “staves” characterize de Groot’s recommended proportions; the inexperienced “staves” characterize the proportions within the formulated eating regimen, normalized to leucine. Notice the intense deficiencies of isoleucine and tryptophan.

Once I regarded on the EAA ratios of the diets that I examined on this manner, it was apparent that the bees might solely absolutely make the most of some fraction of the leucine in every eating regimen, since in each case, another EAA would have been proportionally limiting.

Curious, I spent hours in search of correlations between the EAA ratios within the diets relative to my measured outcomes. I’d be completely satisfied to listen to different interpretations [[9]], however what stood out to me was the sturdy correlation between head & thorax mass on the midpoint, and the sum of the proportional deficiencies in EAAs relative to leucine (Determine 10).

Fig. 10 There was a powerful correlation between head and thorax mass on the midpoint in comparison with the sum of EAA deficiencies relative to leucine (p = 0.0449).

I obtained a good higher correlation after I in contrast whole frames of bees in February (Determine 11).

Fig. 11 There was a strikingly shut correlation between the variety of frames of bees out there for almond pollination in every eating regimen group, relative to the sum of the proportional EAA deficiencies within the diets (p = 0.0048).

For the non-statisticians, the R² (R-squared) worth signifies that 82% of the efficiency of the diets is defined by the impartial variable (the sum of proportional EAA deficiencies). The p worth signifies that the percentages of getting this sturdy a correlation was lower than 5 in 1000. This strongly means that we’re onto one thing vital!

Sensible software: Liebig’s idea of focusing upon limiting vitamins (on this case EAAs) stepwise, didn’t end in the very best correlations. I obtained the very best correlations after I calculated the sum of proportional EAA deficiencies, relative to leucine [[10]]. The take-home appears to be that for greatest efficiency, all the EAA deficiencies add up, so we must always actually attempt for steadiness throughout the board.

Might we certainly predict the efficiency of a man-made eating regimen merely from its ratio of EAAs? De Groot’s analysis suggests sure, as do the outcomes of this trial. Might or not it’s that the EAA ratio relative to leucine is extra vital than having pure pollen as an ingredient, the protein content material or lipid stage, or quantity of sugar within the eating regimen?

Let me level out an fascinating warning from de Groot:

Consideration could also be drawn to the distinct progress which happens throughout the first 7 days on diets devoid of an important amino acid…It’s acknowledged that in our experiments with suboptimal progress charges, one have to be cautious in classifying amino acids the omission of which doesn’t end in a perceptible progress inhibition. These compounds, although not required for progress might exert stimulatory results.

Sensible software: For long-term feeding of a colony (versus the feeding of particular person bees for brief intervals), the “steadiness” between all obligatory vitamins doubtless turns into extra vital. Maybe the addition of pure pollen helps, however maybe it might be pointless if a man-made eating regimen have been higher balanced. Balancing the EAAs seems to be an excellent place to begin!

To that finish, I supply a contribution to our trade…

MY DIET CALCULATOR

As a way to make it simpler for these creating pollen subs, I’ve created an easy-to-use calculator that permits one so as to add elements collectively to create a eating regimen with EAA ratios to match de Groot’s suggestions, normalized to the predominant EAA, leucine, slightly than tryptophan (Determine 12).

Fig. 12 A partial snip of the calculator. Right here’s an instance of what the EAA ratio seems like for a ten:10:5  combination of soy flour, brewer’s yeast, and skim milk powder. Sadly, most commonly-available feedstuffs are quick in isoleucine, threonine, and valine. You may’t see it right here, however the calculator permits one to simply determine how a lot of every of these readily-available amino acid concentrates so as to add so as to steadiness the inexperienced columns to de Groot’s suggestions. The Excel file for the calculator is out there at [[11]].

So for comparability, let’s check out the EAA ratios of a combination of pure pollens [[12]] (Determine 13).

Fig. 13 Not surprisingly (since honey bees and bee-pollinated flowers coevolved), the EAA ratios of a combination of pure pollens (inexperienced columns) intently match de Groot’s suggestions (grey columns).

SO HOW ABOUT THE TESTED DIETS?

Lastly we get to the meat (truly simply the amino acids) of this examine. I used my calculator to create graphs for every of the examined pollen subs (Determine 14).

Fig. 14 For comparability, I positioned the ratios for royal jelly on the high, and positioned the subs roughly so as of efficiency high to backside. Notice how the better-performing subs exhibit fewer gross deficiencies in EAAs. For full utilization of the leucine, each examined eating regimen might doubtless use addition of isoleucine and valine, and a few, threonine.

Since I ran this trial for everybody’s profit, I despatched every producer the calculations for the precise quantities of every EAA so as to add to their formulations to steadiness them out. I’m very curious as as to if doing so can enhance the efficiency of a eating regimen.

Sensible software: I recommend that it’s time for our trade to maneuver past the “by guess and by golly” methodology for formulating pollen subs. Since discipline trials to check efficiency are pricey and require many hives positioned the place there isn’t a pure pollen circulation, formulating diets based mostly upon EAA nutrient mannequin is perhaps an excellent first step. I hope to run trials subsequent season to validate this speculation.

Because the availability of reasonably priced feedstuffs out there for bee diets varies drastically from nation to nation, I designed my calculator to be used by beekeepers worldwide by offering the power to enter the EAA evaluation of anyfeedstuff, in addition to the choice of including EAA concentrates. You may obtain a duplicate of Randy’s Amino Acid Ratio Calculator at https://scientificbeekeeping.com/7333-2/

THE HOMEBREW FORMULATION

Oh sure, plenty of you’re doubtless curious in regards to the elements within the high-performing Homebrew formulation. The beekeepers who offered me the patties helped to fund this mission, however want to stay nameless (I can say that they have been assisted by advisor Alisha Taff [[13]]). Nonetheless, for the advantage of beekeepers all over the place, they’re generously permitting me to share their recipe:

Notice: the above recipe has been corrected from that printed in ABJ.

The beekeepers additionally added a gallon of a seaweed extract from New Zealand [[14]].

NOT YET DONE …

De Groot understood that as monumental as his analysis was, his recommended EAA ratios might not apply to crucial issue concerned in colony buildup — the dietary necessities of the nurse bees for producing the jelly to feed larvae. There can also be separate optimum ratios for the creation of the “storage proteins” obligatory for the well being and longevity of “winter bees.” I’ll handle these topics in my subsequent article.

REFERENCES

[1] de Groot, A. (1953) Protein and amino acid necessities of the honey bee. Physiol. Comp. Oecol. 3, 197–285.

[2] Hagedorn, H (1967) The speed of pollen consumption by newly emerged honeybees. J. Apic. Res. 6, 159–162.

[3] Haydak, M (1934) Adjustments in whole nitrogen content material throughout the lifetime of the imago of the employee honeybee. J. Agr. Analysis 49: 21-28.

[4] Please seek advice from Determine 3 within the earlier (October) installment of this sequence: A comparative trial of the pollen subs Half 4: Why did some subs outperform others?

[5] Altaye, S, et al (2010) Convergence of carbohydrate-biased consumption targets in caged employee honeybees fed completely different protein sources. The Journal of Experimental Biology 213: 3311-3318.

[6] Somerville, D (2001) Dietary worth of bee collected pollens. RIRDC Publication No. 01/047.

[7] McCoy, R. H., Meyer, C. E. & Rose, W. C. (1935) Feeding experiments with mixtures of extremely purified amino acids VIII. Isolation and identification of a brand new important amino acid. J. Biol. Chem. 112: 283–302.

[8] Which requires dividing its proportion by de Groot’s really useful proportion.

[9] The uncooked information is out there at https://scientificbeekeeping.com/7333-2/

[10] The uncooked information is out there at https://scientificbeekeeping.com/7333-2/

[11] https://scientificbeekeeping.com/7333-2/

[12] Taken from de Groot’s Desk 25, which is analogous to different pollen information that I’ve discovered.

[13] https://www.beebiz.internet/

[14] https://agrisea.co.nz/