Monday, August 11, 2014

Swirled or shaken? Does shaking actually damage milk - the scientific evidence


UPDATE: WE HAVE BEEN TOLD THAT THERE ARE SOME ISSUES WITH THE UNITS. WE ARE LOOKING INTO THIS AND WILL UPDATE ASAP - FOR NOW WE HAVE REMOVED ALL CONVERSIONS AND INCLUDE ONLY THE EXACT NUMBERS FROM PUBLISHED STUDIES. EVEN WITH THE CHANGED UNITS, BASED ON THE SIMILAR FINDINGS ELSEWHERE, IT IS UNLIKELY THAT THE CONCLUSIONS WILL CHANGE.

Perhaps one of the most widespread pieces of advice women expressing milk will hear is about the best way to remix milk after expression. Human milk separates after expression (Figure 1) and needs to be remixed before feeding. 

Figure 1: Milk samples (1.5 mL) from 3 different mothers allowed to separate to show the variation in milk fat. Photo: EA Quinn/Biomarkers & Milk.

Many, many websites and books have strict recommendations for the remixing: swirl, never shake. 

As an anthropologist and a bench scientist, I am always interested in the natural history of advice, Where did this advice to swirl, never shake, come from? Upon investigation, I found 3 primary reasons given for why expressed milk should be swirled, never shaken:
1)      Shaking denatures proteins
2)      Swirling helps to remove fat globules stuck to the side of the container
3)      Shaking damages cells.

But, like many before me, I can’t find any actual scientific evidence. I started with PubMed, the national, searchable database of scientific literature ( Figure 2).   

Figure 2: Screenshot of my PubMed search for shaking breast milk. Stirring breast milk looked similar, but with less hits. None were relevant. Image: me.
Here is what I found – and how I went about trying to solve this issue.
Let’s start with #1: shaking denatures proteins. There are many, many different types of proteins in human milk and these are highly variable in size. In addition to size variations, there are also going to be major differences in the way in which proteins are folded – with denaturing being the unfolding of these proteins. 

There are no published papers on this topic. Since the literature was not an option, I turned instead, to math and physics. The idea that shaking denatures proteins is based on the shear force the proteins would be exposed to during shaking.  We need two pieces of information here: what level of force is generated by shaking and what level of force denatures proteins. 

Several reference values for the shear force necessary to denature proteins were available in the literature. Most data however, were based on experimental models of the protein in isolation, when micro-tweezers could be used to literally rip the protein apart. This model is not valid here – what we need is a measure of the shear force necessary to denature a protein in a liquid medium.  Again, we don’t have any studies in human milk, so we will have to substitute water as a medium – and given the composition of human milk, this is a reasonable substitute. In a highly viscous medium, similar to milk, α-amylase (a protein involved in starch digestion found in breast milk), requires a force of 3 x 10^4 Pa to denature the protein.
http://www.rcsb.org/pdb/images/1hny_bio_r_500.jpg
Figure 3: Alpha-amylase, of pancreatic origin. Image from: http://www.rcsb.org/pdb/explore.do?structureId=1hny
Proteins with beta folds, it is estimated, would be much more resistant to shear force. The predicted force (in a highly viscous medium) necessary to shear a beta protein would be 2 x 10^5 to 10^7 Pa. 

So how much force can a human arm generate? Again, there is no direct measurement for a human shaking a highly viscous medium (but there is plenty of data on ketchup).  If you’ve goggled this (or seen Mythbusters) you know an elite boxer can punch with 5000 pounds of force, or more than 22,000 Newtons. 
Figure 3: The action of boxing, as demonstrated by Manny Pacquiao, is very, very different than the action of shaking breast milk in a container. Image: http://thegrio.com/2014/04/13/manny-pacquiao-beats-timonthy-bradley-by-decision-in-boxing-rematch/

But boxing, pitching, and shaking are very different actions – and this causes some interesting differences in the way in which force must be calculated.
When you pitch or punch, the entire body is involved in the action. Punching involves rotation at the waist, shoulder, and elbow. Pitching involves the same rotation, plus the fingers. But shaking is typically done with a stationary shoulder and body and the primary point of movement at the elbow. This is going to limit the force the arm is generating – and the forces extended to the container.  The best analogy in the literature for shaking a container is, remarkably, swinging a hammer, as the hammer swing comes mostly from the elbow. Even a hammer swing is probably an over-estimation, as the shoulder may be involved. 

The average speed for swinging a hammer is 4 meters per second, with maximum times closer to 10 meters per second. The average hammer weights about 3 pounds – the average container of breast milk will weigh a little bit more than 4 ounces.  Now, one thing about a liquid medium is that the forces within the fluid may vary considerably – but it is still unlikely that the human arm will generate enough force through shaking to damage the proteins.  Earlier studies (Thomas and Dunnill 1979) reported that proteins were often stable under shear forces exceeding 9000 s-1 for more than 15 hours. 

One additional factor serves to protect the proteins in human milk, particularly those proteins that are hormones or immune factors rather than more nutritional proteins. We know for example, that many of the hormone proteins are bioactive infant circulation, and thus survive digestion in the infant stomach. Many of these protein hormones are found in a glycosylated form – that is, with the protein has added sugars attached to it that protect the protein structure and serve to reduce the risk of denaturing.  Other proteins may be packaged within the membrane bound fat globules, which will further act to protect the proteins from damage. 

Skipping ahead to #3 – shaking damages cells – the math from above remains important. Again, it is unlikely that the human arm is capable of generating enough force to damage the cells in the milk. Most of the research looking at shear forces and cell damage uses a platelet cell model (Christi 2001). Platelets are not found in human milk, and are also more prone to cell damage and death than many of the other cells commonly found in human milk. Again, human milk specific data are not available – except for spinning in a centrifuge – and we are substituting a leukocyte model for the reference cell. Moazzam et al., (1997), in a study of leukocytes exposed to shear forces in a rat model, found that leukocytes incurred very little damage from shear forces.  Breast milk cells are likely exposed to high shear force at multiple points in their normal life course – from milk ejection to swallowing to digestion, and may be more resistant to cell damage (Papoutsakis 1991). 

Concern #2: Swirling helps remove the fat stuck to the side of the contained.
Again, there are no available data. However, in a study of ultrasonic mixing versus stirring, Garcia-Lara et al., (2013) found that samples mixed by ultrasonic waves had higher fat, suggesting that the ultrasonic mixing was better at removing fat adhering to the sides of the container compared to manual mixing. Current research protocols for measuring milk fat in samples have used multiple inversion techniques to mix milk to ensure adequate mixing – and inversion is a lot closer to shaking than swirling.

So what is the final verdict? There is no published evidence to support that shaking actually damages breast milk when compared to swirling. Many of the issues identified with shaking are better described as myths, and simply do not hold up when the actual shear forces are calculated. Certainly, it would be awesome if we could do an in depth study of this – have women swirl and shake milk with sensors on the hand and in the milk cup and actually measure the acceleration of the hand and then analyze the milk. I suspect however, that we wouldn’t find much damage. 

Sarah and I were discussing the origins of this myth while I was working on this post over the last several days.  She made a really excellent point about this myth – “Really I think it's just one more way to make breastfeeding seem super hard and easy to mess up.” And it seems to be one piece of advice that while well meaning, may contribute to the persistent idea that human milk is fragile, easily damaged, and requires a high degree of care. It serves as one more perceived “threat” mothers (and fathers and caregivers) pose to human milk – the “if you aren’t careful, you’ll damage it and you can’t damage formula*” underlying subtext that serves to undermine breastfeeding mothers.

*see all the recalls and allowable insect parts


References
Bee JS, Stevenson JL, Mehta B, Svitel J, Pollastrini J, Platz R, Freund E, Carpenter JF, Randolph TW. Response of a concentrated monoclonal antibody formulation to high shear. Biotechnol Bioeng. 2009 Aug 1;103(5):936-43. doi: 10.1002/bit.22336.

Yusuf Chisti. Hydrodynamic Damage to Animal Cells Critical Reviews in Biotechnology, 21(2):67–110 (2001).

García-Lara NR, Escuder-Vieco D, García-Algar O, De la Cruz J, Lora D, Pallás-Alonso C. Effect of freezing time on macronutrients and energy content of breastmilk. Breastfeed Med. 2012 Aug;7:295-301. doi: 10.1089/bfm.2011.0079.

Jaspe J, Hagen SJ. Do protein molecules unfold in a simple shear flow? Biophysical Journal. 2006;91(9):3415–3424.

Moazzam F1, DeLano FA, Zweifach BW, Schmid-Schönbein GW. The leukocyte response to fluid stress. Proc Natl Acad Sci U S A. 1997 May 13;94(10):5338-43.

Papoutsakis ET. Fluid-mechanical damage of animal cells in bioreactors. Trends Biotechnol. 1991 Dec;9(12):427-37.

Physics@ UNWA. Smashing bricks and the ballistic pendulum: more collision examples. URL: http://www.animations.physics.unsw.edu.au/jw/smashing-bricks.html. Accessed: 8/9/14.

Thomas CR, Dunnill P. Action of Shear on Enzymes - Studies with Catalase and Urease. Biotechnology and Bioengineering. 1979;21(12):2279–2302.

Thomas CR, Greer D. Effects of shear on proteins in solution. Biotechnology Letters 2010; 33(3) 443-456. DOI : 10.1007/s10529-010-0469-4.

van der Veen ME, van Iersel DG, van der Goot AJ, Boom RM. Shear-induced inactivation of alpha-amylase in a plain shear field. Biotechnology Progress. 2004;20(4):1140–1145.
 

26 comments:

  1. The main reason I had always heard was that swirling doesn't aerate the milk as much as shaking it does. And especially for babies that suffer reflux or gerd you want as little air in the milk as possible, so that burping and regurgitation is limited. There were times when I shook my daughter's milk, and from my own experience, she was definitely more "burpey" than when she nursed or the milk was swirled. :) Just my little anecdote!

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    1. Hi Kay, this certainly sounds plausible, but would also be a concern with shaking formula. We decided to focus very narrowly just on the 3 most common aspects of the myth - with the milk, not the infant, as the outcome measure.

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  2. You need to research bubbles. Shaking causes bubble formation. Bubbles burst and damage biological substances, kill cells, may harm proteins as well. Breastmilk contains cells that will certainly be harmed by bubble formation.

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    1. Hi baharb, we were primarily focused on the aspects of the shaking versus swirling that related to the primary features of the myth - shaking denaturing proteins or damaging cells. As for the bubble formation, based on published papers I have found on cell damage and bubble formation, it looks like bubble formation significant enough to cause cell damage would require shaing in excess of 600 rpm.

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  3. Another blog has published a similar analysis - our conclusions are quite similar. http://www.mypensacoladoula.com/shaken-not-swirled/

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  4. I had heard that the advice to swirl was due to some women having excess lipase in their milk and shaking caused the milk to develop a soapy taste.

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    1. HI Lillibet, we invested the most common manifestation of the debate - we cannot speak to the effect this may have on lipase - hopefully in the future, we will have time to discuss lipase on the blog.

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  5. Well done! This advice also concerned me, as I'd hate to lose fat stuck to the container in order to avoid shaking the milk. That said, another concern I've encountered with shaking is oxidation. My milk turned bitter without going bad very quickly after being expressed and after hunting around for possible reasons, I discovered greater stability in taste when I stopped taking fish oil supplements. The rationale is that high levels of PUFAs made the milk more susceptible to oxidation. Theoretically, shaking the milk would also increase oxidation. I didn't experiment with shaking vs swirling though, and baby seems to do just fine with the bitter milk anyway.

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    1. Again, we don't know - and sadly, I don't see a granting agency lining up to provide research funds to allow us to shake & swirl milk from different donors while using biomechanical imaging that would allow us to calculate the movements, and then analyze the milk for nutrients and hormones. I wish they would - and we would happily look into this question if we could, but the equipment and supply costs (analyzing the milk) are too large for us to investigate this without some form of research support. Maybe we'll do a kickstarter next year once my new lab group is settled and our current batch of projects are reduced.

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  7. Great analysis! Very helpful.

    However, I've always been more concerned about shaking causing bubbles that could cause gas in babies. Do you think this is also a myth? This might be harder to show, since I'm not sure how well the cause for gas is understood

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    1. Hi Griselda, we cannot speak to whether or not shaking may increase gas in infants. Our focus was just on the three aspects of the practice we defined above - and in the case of gas, it would seem that shaking formula might also cause a similar problem. This certainly seems plausible, but was outside the realm of the specific question we were investigating.

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  8. I had understood swirling was recommended because some women have excess lipase in their milk and shaking the milk causes the milk to develop a soapy taste.

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    1. Hi Kathy, we did not investigate this with regards to lipase, only in regards to the denaturing/cell death myths, so our findings cannot contribute to that conversation. I'd love to look into lipase in the future!

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    2. Anecdotally, as a woman with high lipase I've never found shaking vs swirling to make a difference.

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  9. Hi there! I'm that other author. :) This article is incredible, I read it with relish. Thank you for this angle!

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    1. Thanks Jen! I read your piece right after - it was excellent! We went back and forth on whether or not to try and calculate the forces and decided to give it a shot - unnecessary it seems and we have since pulled the calculations, but I thought it was great that we each took a different angle on the question.

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    2. I also loved your historical investigation of where the practice came from - we had not looked into that, and it was awesome.

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  10. From what I have recently read in " Breastfeeding Management for the Clinician" by Marsha Walker, RN, IBCLC - on p. 32 she talks about Vitamin C (ascorbic acid). That the mean concentration decreases over a 20min period of time. That "those bottles with the largest air-to-surface interface had the greatest decrease in mean concentration of ascorbic acid over time. The air moving through the milk and the formation of bubbles on the surface of the milk could be factors in the observed decrease of ascorbic acid concentrations." And that for "those infants who are solely dependent on bottle feeds for their total ascorbic acid intake, especially high-risk or preterm infants...caregivers should avoid shaking the bottle."

    She (the author Marha Walker) cites the following reference: Francis, J., Rogers, K., Brewer, P., Dickton, D., & Pardini, R. (2008). Comparative analysis of ascorbic acid in human milk and infant formula using varied milk delivery systems. International Breastfeeding Journal, 3, 19. Retrieved from http://www.internationalbreastfeedingjournal.com/contnt/3/1/19.

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    1. We did not look at ascorbic acid in the calculations. However, the paper above would seem to apply more to the act of bottle feeding than shaking or swirling, as the paper did not investigate these but rather nipple flow rates.

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  11. Careful with your units! You say "In [water], α-amylase ... requires a force of 3 x 10^4 Pa to denature the protein." I assume that you meant that it requires a "shear stress" of 3 x 10^4 Pa, since the Pascal is a unit of shear stress equal to one Newton per square meter. Because the surface area of the inside of a bottle is no where near 1 m^2, the force required to produce this shear stress is also much lower than 30,000 N. If we assume for simplicity that the area interacting with the milk is a square with 5-cm per side, the area is then 0.0025 m^2 and the minimum force to reach 30kPa would be 75 N, or about 17-lbf. This is much closer to "possible" but still well above what you'd actually expect to see.

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    1. Thanks for the corrections! I am not a physicist by training, and the numbers were checked and rechecked by my group (no physicists) and still off. We have removed the conversions from above, as we received 3 different suggestions on the conversions, each of which had different final numbers, so we have removed the conversions.

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  12. I'm not a scientist, but this makes sense to me! I think about the animal milks that have been a major source of nutrition for humans in various regions, for millenia. Cow's milk, especially, separates when it sits for even a short time. The only other one I've used is goat milk and, although it does not separate in the way that cow's milk does, if you leave some in the fridge in a glass bottle overnight and lift it up to the light, you will see that the top part is more opaque than the bottom part. I was instructed, by the lady I purchased raw goat milk from, to shake it before using it. What I am getting at is that if shaking would damage human milk, it would likely do the same with other mammalian milks. That would mean that people have been damaging one of their greatest food sources all these years.

    I, personally, both swirled and shook frozen breast milk. I only shook it gently, though. In light of the claims that shaking damages it, even if there isn't scientific evidence that was uncovered by this research team, I would encourage moms to shake it gently and swirl it, too.

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  13. But bubbles do denature (unfold) proteins. Soluble proteins (usually) hold their shape because the hydrophobic (water-hating) bits are hidden in the middle of the protein and the hydrophilic (water-liking) bits arrange themselves to the outside. (Gross oversimplification I realise, but still valid). At the air-water interface the water-repelling forces are disrupted and proteins can become unfolded because the forces holding them in their natural shape are absent. You probably need to look through the protein-folding literature to find your answers here, rather than looking at the physical forces involved.

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    1. Hi J. Emerson, we will follow up on these - the next few weeks are the beginning of the term, but I'm looking for a researcher who works with proteins (my own research is more in endocrinology - proteins yes, but less than folding. We'll also need to come up with some thoughts about what the glycosylation may be doing to the proteins. However, if you look at Jen's similar piece, she talks quite a bit on protein denaturing and refolding.

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  14. Also, if I missed your comment I apologize - I am traveling for research and it seems like half the notifications are going to my work email and the other half to my home email, so I may have missed a couple that went to the non-work email addresses. Also, in light of the fact that we received several comments redoing the calculations and each set of calculations were different, we have not released those comments but have edited the piece to reflect the suggestions. The numbers are all being rechecked by a colleague - but she just had a baby last week and is doing her own hands on breastfeeding "research" right now and it may be a bit before we can update the conversions.

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