Monday, August 11, 2014

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


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.
Figure 3: Alpha-amylase, of pancreatic origin. Image from:
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:

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

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: 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.