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Manuel Varela: Who Was Louis Pasteur and what did he have to do with pasteurization?

Oct 1, 2017 by

Related imageAn Interview with Manuel Varela: Who Was Louis Pasteur and what did he have to do with pasteurization?

Michael F. Shaughnessy –

  1. Professor Varela—I would say that almost every day, growing up I heard something about milk being pasteurized. Let’s start at the beginning. Who was Louis Pasteur, and where was he born and how did he first get involved in science and experimentation?

If I may once again be so bold as to make another proclamation, it is this: I am firmly convinced that Prof. Louis Pasteur is the greatest scientific investigator who has ever lived. His scientific discoveries and contributions are legion and legendary. His experimental works have had direct and profound influences on the daily lives of virtually everyone who has ever lived since his discoveries became widely known in the mid-19th century.  Pasteur’s universal influence continues to this day in all circles surrounding our lives.

Louis Pasteur was born on the 27th day of December in the year 1822 in the town of Dole, in France, to parents Jean-Joseph Pasteur (his father) and mother Jeanne-Etiennette Roqui.  Pasteur’s interests in the sciences, research, and discovery began in 1844 while in graduate school as an MS student at the prestigious École Normale Supérieure; while both physics and chemistry courses held his interest, it is in the laboratory setting where Pasteur became fascinated with experiments and was inspired to pursue a career conducting scientific research. In 1847, Pasteur earned his doctorate in the fields of chemistry and physics from the Normale.

2. Now, why and how did he research milk, and what problem was he trying to solve?

Prof. Pasteur came to study milk in an indirect fashion by first studying wine. The story has been told over the generations since Pasteur’s time that, in 1863, Parisian business people came to Pasteur, who was already quite famous by this time, requesting that he investigate their problem with soured wine, which greatly compromised its fine quality. Pasteur’s studies into the sour wine problem led him to propose to the wine makers that they practice ‘pasteurizing’ the wine during the winemaking process.

From this wine-saving outcome, other foods and drink were naturally considered.  Prior to milk pasteurization, the problem of preserving food and drink had long been studied, first with sun-drying foods, smoking, spice additions, salting, pickling, fermentation, and heat sterilizing.  Each of these procedures had their advantages and disadvantages. First, they more or less helped to curb food spoilage, with heat sterilizing, developed by Nicolas Appert, being the most efficient method. The problem with the heat sterilization method, however, was that the favor quality may be severely affected.  Clearly, the strong heat method was not good for certain foods, which may be destroyed beyond repair to be consumed.

Pasteur’s method solved this food-destroying problem by focusing mainly on heating the wine and milk just slightly enough to kill microorganisms that spoiled the liquids without destroying the tastes or qualities and without having to kill all of the microbes in order to do so.

To some critics even the lowered temperatures involved in pasteurization were still too high for them, as they argued that the drinks were too much affected to be desirable for consumption.

Pasteur’s main contribution to the study of milk, however, was his discovery that the microbes were the prime cause of food and drink spoilage.  His pasteurization method was aimed at controlling the contaminating microbes in food and drink.

3. I know that milk basically comes from cows- is it okay to drink it from animals? Or can only animals such as goats and sheep drink parental milk? And how and when did humans start to drink “pasteurized” milk?

Milk is a nutritious substance, filled with lots of ingredients which are thought to be good for the general health of mammals, including humans. Indeed, milk has many good health benefits, such as, for instance, providing protective maternally derived antibodies to infants whose immune systems may not yet be fully developed.  Milk is a good source of protein and good quality carbohydrates, such as lactose, the so-called milk sugar that confers a desirable sweet taste to milk and other milk-based foods.

There has been a long-standing controversy whether one should consume raw milk directly from animals (i.e., unpasteurized milk).  Others argue that one should first pasteurize raw milk prior to its consumption. One the one hand, proponents of raw milk drinking insist that its taste, nutritious quality and safety are all maintained. On the other hand, proponents of pasteurized milk drinking insist that contamination with infectious disease causing microorganisms is all but eliminated, ensuring its safety from both morbidity and mortality. The controversy seems to be rooted in the issues associated with putative costs in terms of adhering to regulations, management, reporting, and in the pasteurization process.

On historical and scientific bases, the consumption of raw unpasteurized milk that might be contaminated with microbial pathogens has been known to be directly responsible for causing infectious disease. For instance, cases of tuberculosis, anthrax, cholera, scarlet fever, staphylococcus, typhoid fever and diphtheria have all been definitively documented as having been transmitted by contaminated raw milk.

More recent work, conducted in our research laboratory, showed that when wild-type Escherichia coli bacteria are cultured in pasteurized milk, susceptibility to antimicrobial agents is reduced for a variety of drugs.  Molecular genetic analysis of this effect showed that the reduced drug susceptibility involved a genetic element on the E. coli chromosome, called the mar locus, an operon that, when turned on, shows enhanced antimicrobial resistance to multiple anti-bacterial agents. It is known that even pathogenic strains of the E. coli harbor the mar locus, as well.

Historically the consumption of pasteurized milk began soon after Pasteur developed his pasteurization methods, especially after the publication of a refined procedure in 1866 by Franz von Soxhlet, a German chemist.  He knew of Pasteur’s work with wine pasteurization and proposed that milk and other drinks be pasteurized, too. Today, not only are beverages like milk, juices, and alcoholic drinks routinely pasteurized, but so too are many other food products, such as yogurt, cheeses, ice creams, pickles, etc.

4. Now what exactly do we mean by pasteurized milk? And how did Louis Pasteur come upon this process?

Pasteur’s work with milk pasteurization arose out of his earlier studies with fermentation in the mid-1850s and with wine in the early 1860s.  Pasteur first studied milk in 1857 when he found that milk sours by turning the milk sugar lactose into lactic acid, in a process we now know as fermentation.  At the time, however, Pasteur had correctly claimed that the milk souring process was performed by the microbes, which he called ferments, contained within the milk.  He was widely criticized, however, for claiming that microbes could do chemistry, a notion now known as biochemistry—living beings partaking in conducting chemical reactions.

Most of Pasteur’s critics held that the milk-souring process was due solely to nonlife-based chemical reactions, independent of the living microbes. We now know that the milk souring process involves microbial metabolism in which the lactose sugar is broken down into glucose and galactose by microbial enzymes, broken down further by oxidation to pyruvate molecules, and then fermented into acidic end-products, like lactic acid, by other microbial fermentation enzymes. Pasteur had been correct all along.

Pasteur had been the first to postulate that microbes conducted biochemistry by souring milk and wine and, importantly, by actually making wine and other alcoholic beverages like spirits, beers, liquors, etc. Out of these newly formed biochemical ideas, Pasteur made the greater conceptual leap that if microbes could make milk and wine sick (spoilage by becoming soured), perhaps microbes could also make people sick.  Hence, Pasteur formulated his amazing historically important hypothesis that microbes can cause infectious disease. The hypothesis became known as the so-called germ theory of disease.

The process of pasteurizing milk is meant to prevent its spoilage. It is not necessarily a sterilization process but rather it is a pathogen-killing one, with many millions of microbes remaining alive. Pasteurization itself involves heating the raw beverage to temperatures that kill microbes which, under normal circumstances grow best at temperatures (37°C, 98.6°F)—the same optimal temperature of most mammals, including humans; such moderate-temperature loving microbes are usually the most efficient pathogens towards humans and other animals. In the past, the pasteurization method involved the batch heating of large quantities of milk to 63°C for about 30 minutes.

In modern times, the so-called flash pasteurization method is practiced.  This process heats the milk to 72 °C as it is passed through thin tubes, but for only a very short time period, about 15 seconds. Another method, called ultra-high-temperature pasteurization heats the milk to temperatures as high as 135 °C, but for only about one second!  This latter pasteurization process is controversial as critics claim the taste of milk is adversely affected by this high-heat method.

A variation of the ultra-high-temperature pasteurization method is the ultra-high-temperature sterilization technique in which all of the microbes in the food or drink are killed by using superheated steam at about 140°C for 1 or 2 seconds. For restaurant connoisseurs who use the little packages containing dairy creamers for their hot beverages, the creamer substances have been ultra-high-temperature sterilized, rather than pasteurized.  These sterilized foods and drink have a relatively longer shelf life.

5. I think he also studied fermentation (which I believe has something to do with grapes) can you clarify?

Pasteur’s first experience with grapes involved also his first research studies, in 1844, as a young scientific investigator. He studied a compound found in grapes, called tartaric acid.  His first major discovery showed, optically speaking, that the tartaric acid crystals formed in mirror-image isomers of each other and rotated the plane of light; and these tartaric acid crystal variant mixtures, he called racemic, which was derived from the Latin term racemus, for grape.

Pasteur studied fermentation from two standpoints. The first being lactic acid fermentation and the second being alcoholic fermentation.

Pasteur’s interest in lactic acid fermentation in 1857 came about due to its convenience with which to study.  Lactic acid was produced in mass quantities from beet juice and as a by-product of alcoholic fermentation. In his experiments, Pasteur showed that a microorganism, nowadays thought to be a bacterium called Streptococcus lactis, when grown in a culture medium containing chalk, which was an alkaline substance, plus sugar and an extract of brewer’s yeast for food, produced lactic acid in large quantities, after an incubation period. In essence, Pasteur was claiming that a living being was undergoing fermentation; that is, a microbe was doing biochemistry by making lactic acid.  The publishing of this work in 1857 has long been considered by historians and scientists alike to be the so-called birth certificate for the establishments of the scientific fields of microbiology and of biochemistry.

Pasteur’s work with alcoholic fermentation showed that yeasts (then called ferments) were actually living organisms and that they themselves could produce the ethanol inherent in the process of alcoholic fermentation.  The traditional process for alcoholic fermentation has historically involved the grapes that you spoke of above, which was a good source of the sugar needed to conduct the process. When Pasteur used grape juice as a source of sugar, he noticed bubbling, indicating that carbon dioxide was being produced.

Furthermore, Pasteur showed experimentally that when his yeast ferments had water, sugar, ammonium salts and trace elements, the yeasts could be coaxed to not only produce alcohol efficiently, but that the yeasts, too, were growing by taking up various elements from the sugar sources.

Pasteur reasoned that the yeast cells may have been producing the wine all along; this led to his formulation of the hypothesis that microbes were conducting chemistry.

Pasteur’s studies of fermentation led also to a phenomenon which biochemists nowadays still appreciate.  The phenomenon is known as the ‘Pasteur effect,’ a term coined by Prof. Otto Warburg, another famous biochemist, in which Warburg meant it to be the inhibition of the glycolytic pathway when in the presence of oxygen. In 1861, Pasteur made the astute observation that oxygen was inhibitory to fermentation. Later, in 1875, he boldly proclaimed that fermentation was life without the necessity for oxygen. Life could occur without oxygen as far as fermentation was concerned. The Pasteur effect is still included in modern biochemistry textbooks.

6. Now, what is the “germ theory of disease” and how is he linked with it?

One of Pasteur’s most famous hypotheses was the idea that microbes caused infectious disease, the so-called germ theory of disease. Interestingly, this theory had its origins with his work on alcoholic fermentation, which, in turn, had its roots when local winery owners approached Pasteur concerning the problem of their vintage wines having gone off, being soured and becoming more like vinegar than wine.  He studied the healthy versus the sick wines with the microscope; in the good wine, he found large yeast cells and in the unhealthy wine, he noticed the yeast cells plus smaller bacterial cells.

Pasteur reasoned that the bacteria were spoiling the wine; this led to his formulation of the famous germ theory of disease.  His reasoning was that if the microbes made wine sick, then perhaps microbes were making people sick, too.  Unfortunately, Pasteur did not do any experimental work to provide any evidence for this germ theory of disease.

The first evidence provided in support of Pasteur’s germ theory of disease was published in the 1870s with Dr. Robert Koch, who studied anthrax disease. Koch developed a methodological approach still in use today, called Koch’s Postulates, in which he showed that a rod-shaped bacterium, now called Bacillus anthracis, was the causative agent of the anthrax disease, thus definitively demonstrating Pasteur’s theory that microbes could cause disease.  Koch was later to demonstrate in 1882 that the bacterium called Mycobacterium tuberculosis, was the causative agent of the dreaded consumption, a discovery for which Koch was to receive the Nobel Prize in 1905.

7. I remember from college the terms “spontaneous generation” which was later disproved- germs, bacteria and viruses- DO NOT just appear out of thin air- How was Louis Pasteur involved in this?

The historical term spontaneous generation has its origins reaching as far back as 343 B.C. with Aristotle, who first proposed the idea in his writings on the topic of the natural sciences. Aristotle wrote that living organisms could arise spontaneously out of non-living materials, like living flies arising spontaneously on non-living rotting meat.  In short, the idea was that life arose out of non-life. The modern term for this phenomenon is abiosis—life from non-life. The abiotic notion of spontaneous generation remained in academic circles for almost two millennia. No one since Aristotle had recorded an experimental examination of spontaneous generation—that is, no one had actually bothered to put the idea to the test—until 1668, when Francesco Redi had published the first experimental analysis of the phenomenon.  Redi’s published evidence argued against the idea of spontaneous generation, at least for flies on meat, that is.

In 1750, John Needham had tested the spontaneous generation in the then newly discovered microorganisms (bacteria) on mutton gravy where he failed to adequately heat the test medium to completion to eliminate contaminating heat-resistant endospores.  Upon cooling, the bacterial endospores germinated and grew to visibility.  Thus, Needham erroneously argued that spontaneous generation occurred in bacteria. This was essentially the state of scientific affairs when Pasteur entered into the arena of spontaneous generation in the early 1860s.

Pasteur’s entry into the detailed study of spontaneous generation started with his curiosity about his studies on milk spoilage into lactic acid, fermentation of sugar into wine alcohol by yeast, and wine spoilage into vinegar. He had established that microbes were responsible for each of these fermentative processes. Next, he contemplated the origin of the microbes involved in these processes.  He had wondered where the microbes came from.

Although Pasteur addressed spontaneous generation by conducting many experiments, two classic ones deserve mention. The first involved grapes and yeast.  He obtained grapes that were free of yeast and showed that they could not ferment, while grapes injected purposefully with the yeast freely underwent fermentation. This work, thus, demonstrated that the yeasts were responsible for conducting the biochemical process of converting the sugar within grapes into alcohol. A comment of Pasteur’s work by the famous physiologist Claude Bernard was made in which he (Bernard) speculated that yeasts underwent spontaneous generation.  This led Pasteur to conduct in the early 1860s what is considered by many to be the most famous scientific experiment ever performed—the so-called swan neck flask experiment.

Pasteur put the spontaneous generation theory directly to the test with microorganisms and the swan neck flask. Pasteur was said to have actually constructed these glass vessels himself.  The swan neck flask has a round bottom with an elongated neck that winds around like the neck of a swan—hence, the name, swan neck flask. The neck of the flask allowed air to move about freely as air was considered the vital driving force for the spontaneous generation process to occur.

A simplified explanation of the famous swan-neck flask experiment follows here.  First, Pasteur filled his swan neck flasks with liquid broth culture media, a so-called yeast infusion broth.  Next, Pasteur heated by boiling the broth-filled swan necked flasks, in order to kill off any microbes contained within the broth—luckily for Pasteur, in the broth there were apparently no confounding heat-resistant bacterial endospores that would have survived the heating.

Non-boiled flasks were included as controls. Next, he allowed the sterile swan neck flasks to incubate, allowing both air and time for any putative microbes to generate spontaneously in the broth and grow. After sufficient time, about 2 or 3 days, there was no growth in the boiled flasks while the unheated flasks had obviously visible microbial growth. Next, he actually broke the necks of the heated flasks without microbes and allowed the microbes from the air to fall into the broken flasks to encounter the sterile culture broth and grow after 1 or 2 days.  Pasteur published these initial findings in a scientific journal on the 6th of February in 1860.

With time, he followed up on these preliminary findings with additional and more-sophisticated experiments, using other culture media like milk and urine and invoking variations in the swan-neck flask technique, but the damage to spontaneous generation had been done. With only a few stubborn holdouts remaining, spontaneous generation was, nonetheless, no more. Spontaneous generation was, as you stated above, disproved finally.

8. I also understand that Pasteur was somehow involved with the rabies vaccine. I understand that being bitten by a rabid dog can result in death. What was his involvement in this realm?

Indeed, throughout most of written history, rabies has been a terribly dreadful disease. Death by rabies is frightening and slow. It is extremely rare to survive rabies, even after a vigorous course of treatment. Only in recent times, has there been a handful of documented cases in which patients survive rabies after infection, as seen, for example, with chemical induction of a coma.

Treatment of rabies in Pasteur’s time was equally frightening. Pasteur had recalled a childhood memory, from 1831, of the terrifying course of treatment for unlucky victims—namely, the cauterization of the flesh wound with a red-hot iron where the rabid animal had bitten the patient. The treatment frequently failed, and the victim often died anyway, usually after having first gone insane.

The recurrence of the dreaded rabies in the region where the grown up Pasteur was living, in 1885, and his success with having developed an anthrax vaccine, compelled him to take up the study of rabies for the ultimate purpose of inventing a rabies vaccine.

First, Pasteur began a series of experiments meant to cultivate the rabies agent; and after a long series of setbacks, he found that rabbit spinal cord was a good medium with which to maintain the rabies microbe. One of the most famous oil paintings of Pasteur is the portrait of him sitting next to his laboratory bench leaning on a book and holding a glass vessel containing a hanging piece of rabbit spinal cord laden with the rabies agent—his is shown looking intensely at his rabies cord. Using this spinal cord method of cultivation, Pasteur was able to isolate and propagate the most virulent deadly form of the rabies agent, testing his virulent rabies on dogs in the laboratory, giving them rabies rather quickly.

Next, Pasteur used this highly virulent rabies strain to begin the process of attenuation—a technique of drying the spinal cords and weakening the rabies microbe in order to lose its virulence but at the same time retain its immunogenic properties, the latter of which were needed to ensure a good, i.e., protective, vaccination. He initially began with the least virulent forms of the rabies on the spinal cords and used increasingly virulent rabies forms to inoculate the laboratory dogs.

After inoculating the dogs with the attenuated rabies vaccine, he was gearing up to test its efficacy by then injecting the dogs with the original highly virulent rabies agent to see if the vaccine protected the dogs.  However, a history-changing event occurred before he could do so.

A little boy named Joseph Meister, a nine-year old at the time, had been bitten and severely injured by a rabid dog, and the prognosis was not good. His mother had known of Pasteur’s rabies work and had made a convincing argument to Pasteur himself to give young Joseph his yet untested rabies.  Only after great trepidation and reluctance, Pasteur agreed to provide the requested rabies vaccine to young Joseph. On the 6th of July, 1885, the treatment had begun of the Meister boy, receiving the same course of inoculations as the dogs had been given, starting with the least to the most virulent but attenuated forms.

The method has since been called the ‘Meister method’ of treatment. The wait-and-see period afterwards was an agonizing one for Pasteur and co-workers, namely Dr. Emile Roux, one of his colleagues who had resigned as a result of having witnessed the medication of an untested vaccine on a child. He later returned to the lab when word of the successes with the vaccine became widely known—Pasteur was never to know how Roux had felt about the incident.

Young Mr. Meister lived to survive the entire ordeal. In fact, he lived to be an elderly gentleman. Throughout the years after the rabies vaccine incident, the Pasteur family had kept touch with the Meister family. As a matter of fact, Pasteur had provided funding for young Meister’s education and even made arrangements to provide employment as gate guard at the Institute of Pasteur in Paris, France. Legend has it that as a guard, Meister had refused admission to German Nazi visitors who wanted to visit the tombs of Pasteur and his wife, Marie Laurent Pasteur, days after the German invasion during World War II. Sadly, shortly after the invasion, Mr. Meister committed suicide at home, apparently because of the guilt he felt for having sent his family away during the war, inaccurately believing they had perished because of him.

9. During his lifetime, Pasteur must have kept very busy- as I hear he also studied Pébrine disease. What exactly is Pébrine disease and what are some of the signs and symptoms and what was Pasteur’s involvement in this?

Pébrine is a devastating disease of silkworms in which they are infected with a parasitic protozoan.  Prior to Pasteur’s involvement with the Pébrine, the disease was a virtual mystery. There were no obvious signs and symptoms—only death of the silkworms. What was known about Pébrine was that it killed the silkworms and, thus, an outbreak of Pébrine nearly destroyed the entire silk industry in France in 1865. When people from the silk industry begged Pasteur to study it, he protested that he didn’t know anything about silkworms, and certainly no one knew what caused the Pébrine. Nevertheless, industry representatives and close friends alike insisted he was just person right for the job of figuring out the cause of the malady—he was a scientist with no preconceived notions and, therefore, he wouldn’t be biased towards or against any misleading paths.

He carefully studied the anatomy and physiology of the silkworms, comparing the healthy versus the diseased worms. It was slow and painstaking work—it took him three years, but he eventually identified two causes, one pertaining to the discovery of the infecting protozoan in the eggs of diseased silkworms, and the other, a pathological condition named flacherie, having to do with poor quality nutrients and the starving silkworm. Pasteur even provided various methods that those in the silk industry could employ in order to eradicate the two maladies. Pasteur is credited with having saved the entire silk industry and preventing the spread of the Pébrine to the rest of Europe.

10. What have I neglected to ask?

Interestingly, historians of science, a prominent one being Dr. René Dubos, who wrote an elegant little biography of Pasteur, frequently state that Pasteur had come very close to discovering antibiotics.  During his studies of the bacterium known to cause anthrax, Bacillus anthracis, Pasteur had observed that when the B. anthracis cultures had been mixed with contaminating soil microbes, probably with bacterial species of the genus Streptomyces, the anthrax bacteria grew less well. The work predicted that some growth-inhibitory substance produced by the soil bacteria was responsible for the growth effects on B. anthracis.  Had Pasteur gone one step further, he may have been the first to discover an antibiotic, instead of Alexander Fleming with his discovery of penicillin in 1928.

Pasteur later was to be a strong proponent of Listerism, which was the use of antiseptic agents during surgery and in the dressings of surgical wounds.

In another less well-known experiment conducted by Pasteur and briefly mentioned in his published 1857 paper on lactic acid fermentation, he had reported on the inhibitory actions of onion and garlic juice on the growth of his famous ferments, which we now know to be yeast microbes. In some of our recent work conducted in our laboratory, we found in the published literature that onion and garlic share a similar bioactive principle called allyl sulfide.  Our results showed that the garlic extract and allyl sulfide inhibits a drug efflux pump, called EmrD-3, from Vibrio cholerae, and that garlic extract made other antibiotics work better in a process called synergy. In our published paper, we were delighted to mention Pasteur’s inspiring work.

On the 19th day of October in 1868, Pasteur suffered a massive stroke. At first, he had been severely debilitated but a few months later, he recovered sufficiently enough to begin walking again. Nevertheless, Pasteur was left with a long-lasting partial paralysis on one side of his body. Again, on the 23rd of October in 1887 Pasteur suffered another stroke. This time he was unable to speak and had to have his son read a scheduled speech for him while Pasteur remained silent. He did manage to recover, but he did suffer from partial paralysis for the remainder of his life. He continued to work hard despite his condition, as he had done so all of his life. On the 25th of September in 1895 he passed away, leaving behind an overabundance of amazing discoveries, any one of which in their own right could easily have made him a well-respected and famous scientist.

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