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.
 |
| 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
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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
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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.
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