Active Cultures Add Function to Yogurt and Other Foods

Yogurt was introduced to the American diet during the 1940s. By the 1980s, it had become the darling of dieters, and the lunch of choice for young women. The use of yogurt as a calcium source has made it one of the most rapidly growing products in the dairy case. But now it’s more than just a calcium source. Yogurt, kefir, and similar fermented milk products are on the way to becoming major nutraceuticals, aimed at treating a variety of disease conditions.

There are many products in the marketplace, and more on the development benches. Many can be considered medical foods, some are fortified foods, and others are “healthy” foods. In many cases, the boundaries between the categories are fuzzy and may only exist in the eye of the beholder. 

Organisms involved with probiotic activity in yogurt and other dairy products are varied, and of two specific types: bacteria that are friendly to the lower intestine, and antibodies from milk or colostrum that are effective in disabling pathogens. The organisms used to produce yogurt and similar milk-based products provide function as well as health attributes: they manufacture lactic acid bacteria that disable pathogens, but they are deleterious to the product’s flavor and textural characteristics. 

The variety of these organisms offer different flavor profiles and textural characteristics. Some of the specific organisms are patented, others provide typical flavors of a given product. In recent years, use of several different types of organisms has found favor with companies: the wider variety of organisms is thought to provide more advantages to the consumer. 

U.S. Dept. of Agriculture researcher John B. Luchansky, a patentor in this field, has described the role of various organisms as a continuum (from probiotics to prebiotics and nutribiotics) and has related the use of these biologically active ingredients in foods to their use in animal feeding, where they promote animal well-being and food safety. This continuum is seen in the use of biologically active products in nutraceutical and/or functional foods for the human population. Currently, the primary use of probiotics is in fermented dairy products, but it is unlikely that this will remain the case. 

Prebiotic products used in yogurt and similar products include: Lactobacillus delbrueckii subsp bulgaricus, used for flavor formation, development of lactic acid, rapid acidification; Lactobacillus helveticus, used for rapid production of lactic acid and fermentation of specific sugars; Lactobacillus fermentum, Lactobacillus leichmannii, Lactobacillus plantatum, Lactobacillus cellobiosus, and Lactobacillus casei, which produces less lactic acid; Lactobacillus johnsonii, an organism that has been patented by Nestlé; Lactobacillus acidophilus ER 2, described as flavin producers and bacterial inhibitors; Lactobacillus acidophilus NV, which produces antibiotic activity; Streptococcus salivaricus subsp thermophilus; Lactococcus lactis subsp lacti, which produces nisin; Bifidobacterium infantis, described in use patents assigned to Nestlé; Bifidobacterium longum, Bifidobacterium breve, and Lactobacillus reuteri, which is patented by Biogaia Biologics AB, Stockholm, Sweden. 

Yogurt is made by culturing milk with the organisms, adding thickeners, especially when the milk used is nonfat or low fat, and flavoring the mass with sweeteners, fruit preparations, and a variety of other flavors. Chocolate flavors have been a challenge; nevertheless, a number of companies have developed yogurts with chocolate additions or flavors. The thickeners may be modified starches, hydrocolloids, or pectins, selected to provide a specific texture. Vitamins and minerals may be added, and often are for products given to children. Yogurts can be made from soy milk, thus incorporating phytochemicals, and may contain a source of omega-3 fatty acids or other specialty fats. They may also contain prebiotics, such as fructooligosaccharides or inulin, carbohydrates that provide a good medium for the culturing organisms to multiply in. Yogurts may be spoonable or drinkable, and may be considered dietary supplements for infant consumption. So they cross the line between dietary supplements, medical foods, and conventional foods with great facility. 

But other products besides yogurt have been developed using culture organisms, and more are likely to follow. These include beverages, infant formula, bars, capsules, cheese, and frozen yogurts that may greatly resemble ice creams. 

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Specific Probiotics
The major difference between products, in addition to the amount and kind of supplementation, is the specific organism used as a probiotic. A number of companies and universities have done extensive research on the value of certain culture organisms:
• New Bifidobacterium Strain Acts as a Probiotic. The Wisconsin Alumni Research Foundation has been assigned U.S. patents 5,922,375 and 5,902,743, disclosing a Bifidobacterium strain isolate (ATCC 202078.) The strain, called Bifidobacterium longum JBL 28-1/3300, was defined by DNA testing and is produced in vivo, using human breast milk. By providing the bacterium as a supplement, the protective activity of the Bifidobacterium found in human milk can be produced in suitable foods, including infant formula, fluid and fermented milks, fruit juices, and sports drinks. The bacterium acts as a probiotic, and can assist newborns in producing protective acetic acid and lactic acid, as well as antimicrobials and vitamins. It can also be used to reseed bacteria levels caused by diarrhea, chemotherapy, advancing age, antibiotics, or other causes.

• Streptococcus thermophilus Stabilizes Frozen Yogurt Cultures. Mix for frozen yogurts should have a lower acid flavor for consumer acceptance, but shelf life can’t be affected by Gram-negative bacteria survival that is permitted by lower acid production. As disclosed in U.S. patent 5,910,329, assigned to Rhodia, Inc., by using S. thermophilus to replace L. bulgaricus, which produces more lactic acid, the flavor is improved because of the reduced amount of acid. Viscosity is also improved because S. thermophilus produces polysaccharides during culturing, providing more viscosity and a better texture. S. thermophilus is frequently cultured with L. bulgaricus to prevent the development of lactic acid after yogurt is made, and to improve long-term flavor during refrigerated shelf life of the yogurt products. 

Bacteriocins produced from S. thermophilus are described in U.S. patent 5,683,890, assigned to Nestec S.A. A bacteriocin is a peptide that has antibacterial activity, usually with a fairly narrow activity spectrum. The patent defines the various bacteriocins, plus two new ones with defined amino acid sequence.

• L. reuteri Treats Side Effects of Antibiotic Treatment. Reuterin, described in U.S. patent 5,439,678, is an antibiotic substance produced by cultivating strains of L. reuteri under defined controlled conditions. The strains that are effective in producing reuterin are those that showed inhibited growth in the presence of glycerol or glyceraldehyde: the antibiotic substance is not a polypeptide like bacteriocin, so it is not affected by proteases.

L. reuteri is being used in yogurt marketed by Stonyfield Farm along with four other probiotic products. While the organism is licensed by Stonyfield from Biogaia Biologics for use in yogurt, it is also offered for other applications, such as a powdered nutritional formula for infants or for persons who have been treated with antibiotics and have developed either infectious diarrhea or diarrhea caused by antibiotic reduction in the normal intestinal flora. A patent on the use of L. reuteri was assigned to Biogaia, and a use patent (U.S. patent 5,902,578) that employs L. reuteri, L. acidophilus, and B. infantis in a complete formula or as a supplement was assigned to Abbott Laboratories. Children living in Mexico City were randomized to receive one of two products in a 16-week, blind, parallel feeding trial. The study showed that children who received the probiotic “cocktail” had significantly reduced incidence of diarrhea.

• L. johnsonii cncmi-1225 Fights Pathogens. Taxonomic classification of L. acidophilus was reorganized under the Budapest treaty on June 30, 1992, to include six subgroups, one of which is L. johnsonii. This specific subspecies was described by researchers from Nestec S.A. as a microorganism that gave rise to L(+) and D(–) lactic acid. This particular organism was found to be particularly effective in increasing the phagocytic power of leucocytes of peripheral blood of volunteers during a clinical trial. According to U.S. patent 5,603,930, assigned to Nestec S.A., the organisms adhere to Caco-2 cells, and inhibit adhesion to the blood cells of enterovirulent and enteroinvasive pathogens. 

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Antibodies from Colostrum
Use of antibodies is a recent innovation, triggered by the discovery that colostrum, the first milk of cows that is usually fed to baby calves, carries an immune factor (IgG) that can be utilized in food products. Studies have suggested that antibodies from colostrum are able to neutralize Clostridium difficile, the cause of infectious diarrhea in hospital patients. 

Galagen Inc. gathers the colostrum from Land O’ Lakes’ 750,000 cows during the first day or two after the birth of calves, when colostrum is produced instead of conventional milk. After the newborn calf is fed, the colostrum is collected and concentrated under processes that keep the IgG intact and useful. The process is geared to pharmaceutical standards, which produces a standardized product. The product is dried and the IgG content assayed. Because the product is extremely concentrated, it can be used in products without imparting a dairy flavor. The concentrate tastes rather like a dairy creamer. 

Flavor Enhancement
Yogurt and similar products have come a long way since their introduction in the mid-1940s, at least in preference by the American palate. Consumers accustomed to the particular flavor produced by full-fat yogurt and simple fermentations, sans flavoring agents, frequently rejected the American yogurts, which became lighter in fat content, more heavily flavored with fruit and other flavors, and milder. Use of specific cultures, as mentioned above, can balance flavors and improve shelf life while retaining active cultures. 

The emphasis on lower-fat products is the subject of a great deal of research. Information from U.S. patent 5,6443,621, assigned to Kraft Foods, discloses the use of beta-glycans (material from the cell walls of yeast) as a fat mimetic or a carrier for flavor precursors. A small amount of material derived from butterfat is said to be useful in making low-fat products taste more like full-fat products. Yeast cell wall material is suspended in warm milk, mixed with a cooled liquid butterfat fraction, and mixed into the vat milk. Then the process of making yogurt or cheese can continue in the normal way. 

Kraft has also been instrumental in developing ways to introduce chocolate into yogurt. Generally, yogurt cultures provide an acid note that is incompatible with chocolate. Methods of reducing the acid flavor include use of buffering salts, which can affect pH, so that bacterial stability may be compromised, or heating the product, which reduces the active cultures. Sugar doesn’t do the job, since it simply adds sweetness but doesn’t mask the acidity. By developing a separate chocolate flavoring packet that the consumer stirs in, the active cultures are retained. This technology is described in U.S. patent 6,068,865. Kraft has also worked with nisin-containing whey in formulations of dairy products to prevent development of excessive acid, stabilizing products over the required shelf life. U.S. patent 6,136,351 describes the effect of nisin-containing whey in yogurt products and suggests that chocolate-flavored products may be stabilized against development of off-flavors. 

In most cases, companies developing new yogurt or other fermented dairy products use cultures developed and purchased from supplier companies. Some companies, such as Nestlé, have patented specific cultures, while many companies have done extensive research on the specific cultures and their use. 

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Calcium-Fortified Yogurt
Fortification has become a major method of increasing calcium in the diet. Most consumers know that milk-based foods contain calcium, but they usually underestimate the amount of dairy-based products necessary for a suitable intake. Adding calcium is tricky—solubility and bioavailability are factors. The fermented milk base ordinarily cannot be sheared very much, or the cultures will be damaged. So getting the calcium salts into the yogurt has been a problem. But some new methods of applying additional calcium have been published in patents:
U.S. patent 5,820,903 describes a method of increasing calcium content using a calcium phosphate salt with a reduced particle size. With calcium fortification, yogurt with total calcium content of 500–1,500 mg/170 g can be produced. The calcium phosphate has a particle size of less than 150 μm in the product. The insoluble salt is mixed with a yogurt base after fermentation, using minimal shear. Viscosity of the fermented base is 1,500 cP/sec at 5ºC. A concentrated slurry of calcium phosphate having a pH of 4.0–4.6 is prepared, using an edible organic or mineral acid. The slurry can be homogenized to reduce the particle size. Sufficient amounts of the slurry are mixed with the yogurt base to provide the desired levels of calcium (native plus supplemental). This patent was assigned to General Mills, Inc., and uses their basic knowledge about calcium and yogurt products. 

Earlier technology described in U.S. patent 45,449,523, assigned to the Ohio State University Research Foundation, notes that practical difficulties encountered in adding calcium to dairy products is caused by the very low solubility of calcium salts in milk. Of the calcium in milk, 60–70% exists as insoluble colloidal calcium phosphate associated with the casein micelles. Calcium salts, being generally insoluble and not suspended by casein, settle out so that uniform dispersions during manufacture are not achieved. Suspending insoluble calcium salts is complicated because the yogurt base should not be agitated during the incubation period. During vat incubation, a substantial portion of calcium salt settles to the bottom of the vat and is scraped off and blended into the yogurt. In light of these cleaning difficulties, conventional yogurt manufacturing equipment makes providing calcium-enriched product difficult—and blending calcium into the product may affect the texture. 

The patent shows that a higher-calcium product can be produced by making a yogurt base mix which includes at least one fermentable dairy ingredient and a calcium source, plus potassium hydroxide and sodium citrate added in amounts effective to prevent the pH of the base mix from dropping below about 6.7 prior to pasteurization, then pasteurizing the mix, optionally homogenizing it, cooling it, inoculating it with a yogurt starter culture at about 100–115ºF, and incubating it until the pH of the base is reduced to about 3.5–5.0. A calcium-fortified yogurt with 80.3% of the Recommended Daily Intake for calcium was prepared in one example in the patent, without chelating agents, which can also be used.

Looking Ahead
There are now products with complete supplementation offered as medical foods, as well as healthy products for people who have problems obtaining all of the nutrients they need. It is clear from some of the literature that fermented milk foods containing various nutrients are being tested as curatives for specific diseases. 

For instance, research from the Federal Research Centre for Nutrition, Karlsruhe, Germany, and the Dept. of Food Science and Technology at Oregon State University, published in the January 1999 issue of Journal of Nutrition, discussed the role of lactic acid–producing bacteria in preventing carcinogen-induced preneoplastic lesions and tumors in rat colon. 

A number of patents and papers suggest that yogurt and similar foods with active cultures and other ingredients have approached medical food effectiveness in conventional food format. The large number of papers in the literature that describe the clinical trials of these products suggests that products containing active cultures and possibly other forms of disease resistance will continue to be introduced to the food supply.

by Fran Katz
Editor