Targeted cultivation of gut microbiota: from live Bifidobacterium preparations to precise oligosaccharide energy supply

L.M. Mechnikov, the Russian-born Nobel laureate of 1908, believed that aging was caused by toxins from intestinal bacteria, and that eating yogurt could effectively prevent aging. He attributed the high number of centenarians in the Balkans to their regular consumption of yogurt. This became known as Mechnikov's theory of longevity. Later, it was discovered that the main bacteria in yogurt are killed in the acidic conditions of the stomach after oral ingestion, making it impossible for them to "settle" in the digestive organs. Therefore, this theory was gradually forgotten. However, until recently, with advancements in research on intestinal bacteria, the role of beneficial bacteria in the gut has been gradually clarified.

I rediscovered the importance of Mechnikov's theory on intestinal bacteria. While fermented foods like yogurt certainly contain many microorganisms, bacteria are also abundant in ordinary foods. It's generally believed that around 100,000 ordinary bacteria per gram of food is a safe level. The mouth also contains many bacteria; on average, there are about 10 million bacteria per milliliter of saliva. When food enters the stomach, gastric acid begins to be secreted. Gastric acid is hydrochloric acid with a pH of 2, possessing a strong acidity capable of dissolving zinc, thus killing most microorganisms.

This reduces the number of microorganisms to below 1000 per milliliter of gastric juice. When stomach contents are transported to the duodenum, they are neutralized by pancreatic juice and then digested and absorbed in the small intestine. Bacteria are fewer in the upper part of the small intestine, but increase dramatically in the lower part. Undigested food is transported to the large intestine, where the number of microorganisms increases dramatically. When people think of intestinal bacteria, they usually think of *E. coli*. However, surprisingly, *E. coli* is not the most abundant bacteria in the large intestine.

When *E. coli* was discovered, bacterial culture techniques were not yet mature, making it impossible to find the anaerobic bacteria that predominated in the large intestine, as these bacteria cannot reproduce under aerobic conditions. The discoverers of *E. coli* believed this bacterium was the "protagonist" of the large intestine and thus named it *E. coli*. Breastfed infants are less prone to illness than non-breastfed infants, and it was believed that one major reason might be the higher concentration of Bifidobacteria in the intestines of breastfed infants. Therefore, Bifidobacteria attracted considerable attention. The term "Bifidobacteria" implies that the bacteria are branched, or form a "Y" or "V" shape.

From adulthood to middle age, although the composition of Bifidobacteria in the gut changes, their dominance remains constant, and their numbers are maintained at a certain level. However, once old age arrives, the composition of gut bacteria changes again, and some elderly people even have no Bifidobacteria at all in their intestines. Conversely, the number of harmful bacteria such as Clostridium increases with age. This indicates that the intestinal system of the elderly or infirm has degenerated, making it no longer possible for Bifidobacteria to survive. Clostridium, Escherichia coli, and other bacteria commonly present in the intestine can utilize food to produce putrefactive substances such as ammonia, amines, indole, phenols, nitrosamines, and hydrogen sulfide.

In contrast, beneficial Bifidobacteria do not produce substances such as ammonia, amines, or phenols. Studies have shown that feeding animals Bifidobacteria can enhance their immunity. Many studies have also shown that Bifidobacteria are beneficial for the elderly, infants, and pregnant women. Furthermore, Bifidobacteria play a role in synthesizing essential B vitamins and other vitamins in the intestines. B vitamins are essential for preventing fatigue. Additionally, VB₂ and VB₆ are also skin vitamins. Therefore, an increase in intestinal Bifidobacteria can prevent rough skin and maintain its moisture.

Vitamin B₁₂ is crucial for preventing anemia. Therefore, individuals experiencing poor complexion, menstrual irregularities, coldness, dizziness, or other symptoms should adjust their diet and lifestyle to increase bifidobacteria in the gut. Furthermore, among the vitamins synthesized in the intestines, besides B vitamins, vitamin K is also very important. A deficiency in vitamin K makes it difficult to stop bleeding due to injury or other causes, and gums are also prone to bleeding. Based on these reasons, it can be concluded that oral administration of live bifidobacteria is very effective in maintaining health. This effect has indeed been proven in practice.

Furthermore, milk contains nutrients necessary for the growth of Bifidobacteria, making it beneficial to consume Bifidobacteria along with milk or dairy products. Bifidobacteria are anaerobic bacteria and cannot survive in aerobic conditions, making them difficult to utilize in food. However, strains that can survive in aerobic conditions have been obtained in recent years. Bifidobacteria can produce equal amounts of acetic acid and lactic acid. Therefore, when using Bifidobacteria to make yogurt, it will have a pungent acetic acid odor, making it impossible to produce delicious yogurt.

Methods to inhibit acetic acid production and techniques to mask its odor have been developed, leading to the availability of a wide variety of delicious Bifidobacterium products. Furthermore, many of these products also include added beneficial ingredients such as calcium. Ingested Bifidobacteria repeatedly multiply and die in the body, taking approximately one week to be excreted. During this time, Bifidobacteria exert beneficial effects on the health of the consumer. It is unrealistic to expect Bifidobacteria to permanently multiply in the intestines after only one consumption; therefore, daily intake is recommended.

Oligosaccharides, also known as oligosaccharides, have the effects of low calorie value, preventing tooth decay, and promoting the proliferation of beneficial bacteria in the intestine. The benefits of Bifidobacteria proliferation in the intestine have been described above. In order to promote the proliferation of Bifidobacteria in the intestine, it can be directly ingested as mentioned above. In addition, there are other methods, namely, ingesting nutrients for Bifidobacteria to fundamentally help the proliferation of Bifidobacteria in the intestine. As a nutrient source for Bifidobacteria, the following conditions must be met: (1) Bifidobacteria can utilize it; (2) It is not digested and absorbed in the human stomach and small intestine and can reach the large intestine; (3) Harmful putrefactive bacteria in the intestine, such as Persicae, are difficult to utilize it.

It has been discovered that several oligosaccharides can meet all these conditions. Oligosaccharides, as opposed to polysaccharides, are composed of 2 to 10 monosaccharides and are mostly sweet. Disaccharides are composed of two monosaccharides (such as sucrose and maltose), trisaccharides are composed of three monosaccharides (such as maltotriose), and so on, including tetrasaccharides, pentoses, and so on. Starch and cellulose, on the other hand, are composed of many monosaccharides and are therefore called polysaccharides. Among polysaccharides, starch can be digested and absorbed by digestive enzymes to become a source of energy. Cellulose, however, cannot be digested and belongs to the category of dietary fiber.

Oligosaccharides include those that can be digested and used as a source of energy, like sucrose, and those that cannot. Substances that can serve as a nutrient source for bifidobacteria in the gut must be indigestible, because if they could be digested and completely absorbed by digestive enzymes, they would never reach the large intestine. Fructose-oligosaccharides (FIOs) were the first commercially available oligosaccharides that could increase bifidobacteria. Meiji Confectionery Co., Ltd. developed the industrial production of FIOs. FIOs are formed by binding 1 to 3 fructose molecules to sucrose molecules. Many natural foods, such as asparagus, burdock, onions, garlic, and soybeans, contain low concentrations of FIOs.

Fructose-oligosaccharides (FOS) not only promote the growth of bifidobacteria in the gut, but also do not cause diseases like diabetes, obesity, and arteriosclerosis like sucrose, nor do they cause tooth decay. Because FOS retains the sweetness of sucrose, it has excellent food processing properties. The industrial production method for FOS involves the action of fructosyltransferase on sucrose. This enzyme is extracted from Aspergillus niger. Ingesting FOS can make bifidobacteria dominant in the gut, improve constipation, and also improve lipid levels in patients with hyperlipidemia. However, some harmful bacteria in the gut, such as Escherichia coli and Laminaria japonica, cannot utilize FOS.

Fructose oligosaccharides are not only beneficial to humans, but also have excellent effects on pets, piglets, and other livestock. In the case of pigs, piglets are weaned at about one month old. During this period, piglets no longer receive immunity from their sows and enter a stage of developing their own immunity. Therefore, diarrhea is common in piglets during this period. Once diarrhea occurs, piglets' weight gain slows down, and their development is delayed. If 0.1% fructooligosaccharides are added to the pig feed during this period, the incidence of diarrhea is significantly reduced compared to feeding regular feed.

Furthermore, the levels of putrefactive substances such as ammonia and skatole in piglet feces are significantly reduced, leading to a marked improvement in weight gain and feed utilization. Adding fructooligosaccharides (FOS) to pet food such as dog and cat diets reduces constipation and diarrhea, and also lowers fecal odor. FOS is not only low in calories, but unlike sucrose, it does not cause tooth decay. Because FOS shares physical similarities with sucrose, it can be used as a substitute, offering a similar taste and making it suitable for manufacturing low-calorie cookies, ice cream, and other similar foods.

Food products made by combining Bifidobacteria and fructooligosaccharides are already commercially available. Soybean oligosaccharides are a general term for oligosaccharides found in soybeans and are a natural food that humans can consume long-term. The structure of soybean oligosaccharides consists of glucose and fructose with one or two galactose ions attached. A Japanese food industrial company, using soybean meal as raw material, became the first in the world to successfully refine high-purity oligosaccharides and commercialize them. This soybean oligosaccharide has a sweetness level of 70% that of sucrose, possessing a sweet and refreshing characteristic. Because it is not easily broken down by digestive enzymes, its caloric value is only about half that of sucrose.

This type of sugar is as stable to acid and heat as sucrose, making it suitable for use in various processed foods. Soy oligosaccharides also promote the growth of Bifidobacteria. Moreover, compared to existing oligosaccharides, only a small amount is needed to increase intestinal Bifidobacteria. Soy oligosaccharides can be specifically utilized by intestinal Bifidobacteria, while harmful bacteria such as *Pericarpium Wechslerae* and *Escherichia coli* cannot. Isomaltose, 6-α-glucosylmaltose, and isomalttriose are all branched oligosaccharides. Because these sugars cannot be fermented by yeast, they are also called non-fermentable sugars.

Oligosaccharides are found in traditional fermented foods such as sake, cooking wine, soybean paste, and soy sauce, as well as in natural foods like honey. These oligosaccharides not only taste good but also enhance the umami flavor of food, retain moisture, act as preservatives, and prevent starch retrogradation, thus improving food quality and shelf life. Isomalutose promotes the growth of bifidobacteria in the intestines and helps prevent tooth decay. Showa Sangyo Co., Ltd. established a large-scale production process for isomalutose in 1985, and this product is now used in various foods such as beverages, candies, cookies, bread, and boiled beans.

Isomerized lactose is obtained by reacting lactose with an alkali. Isomerized lactose is hardly digested in the small intestine, but it promotes the proliferation of bifidobacteria in the large intestine, while also generating large amounts of vitamins B₁ and B₂. If isomerized lactose is given to non-breastfed infants, their feces will contain almost entirely bifidobacteria, similar to the situation in breastfed infants. Isomerized lactose promotes the proliferation of bifidobacteria in the intestine, and as the intestinal pH becomes more acidic, the growth of putrefactive bacteria and pathogens is inhibited, improving constipation or diarrhea and promoting protein digestion and absorption.

In addition, two or three scholars reported at the Japanese Cancer Society that isomerized lactose has anti-cancer effects. Isomerized lactose can be used as a food ingredient in beverages, candies, flavorings, baby food, weight-loss foods, health foods, low-calorie foods, and more. Nikken Chemical Co., Ltd. has attracted attention by marketing isomerized lactose under this brand name.