Agency Response Letter GRAS Notice No. GRN 000429

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CFSAN/Office of Food Additive Safety

December 10, 2012

James T. Heimbach, Ph.D
JHeimbach, LLC
923 Water St., P.O. Box 66
Port Royal, VA 22535

Re: GRAS Notice No. GRN 000429

Dear Dr. Heimbach:

The Food and Drug Administration (FDA) is responding to the notice, dated March 20, 2012, that you submitted on behalf of Yakult Honsha Co., Ltd. (herein after referred to as Yakult) in accordance with the agency’s proposed regulation, proposed 21 CFR 170.36 (62 FR 18938; April 17, 1997; Substances Generally Recognized as Safe (GRAS); the GRAS proposal). FDA received this notice on March 27, 2012, filed it on April 10, 2012, and designated it as GRN No. 000429.

The subject of the notice is Lactobacillus casei (L. casei) strain Shirota. The notice informs FDA of the view of Yakult that L. casei strain Shirota is GRAS, through scientific procedures, for use as an ingredient in fermented dairy products at a maximum level of 4x108 colony forming units (cfu) per milliliter (ml).

As part of its notice, Yakult includes the report of a panel of individuals (Yakult’s GRAS panel) that evaluated the data and information that are the basis for Yakult’s GRAS determination. Yakult considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Yakult’s GRAS panel discusses the identity, phenotypic and genotypic properties of the microorganism, production of the organism, and intended use. They also estimated the dietary exposure, and oral toxicity studies in animals as well as in healthy or compromised adults and children. Based on this review, Yakult’s GRAS panel concluded that L. casei strain Shirota is GRAS under the conditions of its intended use.

Our use of “L. casei strain Shirota” in this letter should not be considered an endorsement or recommendation of that term as an appropriate common or usual name for the purpose of declaring the substance in the ingredient statement of foods that contain that ingredient. Title 21 CFR 101.4 states that each ingredient must be declared by its common or usual name. In addition, 21 CFR 102.5 outlines general principles to use when establishing common or usual names for nonstandardized foods. Issues associated with labeling and the appropriate common or usual name of a food are the responsibility of the Office of Nutrition, Labeling, and Dietary Supplements (ONLDS) in the Center for Food Safety and Applied Nutrition (CFSAN).

Yakult describes the isolation, taxonomy and characterization of L. casei strain Shirota. Yakult describes L. casei strain Shirota cells as rod-shaped (0.4-0.6 x 2-3 micrometers) that occur singly, in pairs, or in short chains comprising three or four cells. The strain is capable of growing at temperatures between 27 degrees Celsius (°C) and 43°C with an optimum of 37°C. While it can grow at a pH as low as 3.5, its optimal pH is 6.5. The strain is a Gram-positive, facultative homofermentative lactic acid bacterium. Additionally, L. casei ferments lactic acid from many other precursors. Only the L-enantiomer of lactic acid is formed.

The 16S rRNA gene sequence of L. casei strain Shirota is 100 percent identical to those of L. casei strains designated ATCC 334 and NCDO 151. L. casei contains no genes encoding for antibiotic resistance and there are no known sequences showing significant homology with known antibiotic resistance genes. L. casei strain Shirota, like most lactobacilli, is resistant to vancomycin; however, this resistance is not genetically based.

Yakult describes the production of L. casei strain Shirota from a renewable production master stock that is inoculated in the liquid culture medium, grown at 37°C until the culture reaches the stationary phase, and then lyophilized and stored at -20°C. Live L. casei strain Shirota master stock is added to the tank containing the fermentation media and allowed to incubate at 37°C for up to a week. The ensuing fermented milk curd is homogenized, and the cultured solution is transferred to a storage tank. Permitted food-grade syrup and flavors are then added and the resulting solution is mixed at approximately 10°C. Prior to bottling, the concentration of L. casei strain Shirota is measured and adjusted to the desired level. The final product is stored at 10°C after testing for organoleptic quality, specific gravity, Brix, titratable acidity, heavy metals and microbiological purity. Yakult states that fermentation of L. casei strain Shirota is carried out in a controlled environment, and in the presence of food grade growth media, under conditions safe for human consumption.

Yakult states that the specifications for L. casei strain Shirota, include L. casei strain Shirota activity, color, acidity, limits for heavy metals including arsenic, cadmium, lead, mercury (all less than 0.1 milligram per kilogram) and microbiologicals including Bacillus cereus, Escherichia coli, Enterobacteriaceae, Staphylococci, molds, yeast, and coliforms (all less than 10 cfu per ml), Listeria species, and Salmonella species (both negative in 25 ml). In addition, Yakult discusses information on stability and shelf life of L. casei strain Shirota for the intended use levels.

Yakult uses consumption data from the United States Department of Agriculture Continuing Surveys of Food Intakes by Individuals and the Reference Amount Customarily Consumed for yogurt to calculate a maximum estimated daily intake for L. casei strain Shirota from the intended uses to be 8x1010 cfu.

Yakult describes L. casei strain Shirota as a non-pathogenic and non-toxigenic bacterium. Yakult states that the complete genome of L. casei strain Shirota has been sequenced, and concludes that there is no evidence of any safety hazard from these data. Yakult discusses the safety of L. casei strain Shirota based on published and unpublished studies including in vitro methods, genetic sequencing, animal models, and controlled trials in human subjects.

The notifier discusses published in vitro studies that demonstrate L. casei strain Shirota’s inability to: adhere to intestinal cells, degrade mucin, produce undesirable metabolites, cause infections, transfer antibiotic resistance genes, or affect the bioavailability of non-steroidal anti-inflammatory drugs. The notifier also summarizes several published and unpublished studies in a variety of animals (mice, rats, hamsters and rabbits) to demonstrate safety. The animal studies have included doses as high as 2x109 cfu/day in mice and 1011 cfu/day in rats, for periods up to 6 months with no adverse effects reported.

The notifier discusses several human studies conducted in healthy or compromised adults and children. The notifier states that L. casei strain Shirota up to 3.6x1011 cfu/day fed to healthy adults for as long as 5 weeks and to health-compromised adults up to 8x1011 cfu/day for as long as four years, showed no adverse effects. Similarly, studies in both healthy children and health-compromised infants fed L. casei strain Shirota up to 3x1010 cfu/day, for as long as 55 months, reported no adverse effects. Additionally, the notifier describes four published case studies in which doses of L. casei strain Shirota (up to 4.5x109 cfu/day) were administered therapeutically to severely compromised infants for as long as 2 years with no indication of adverse effects. The notifier states that these published studies are supported by three unpublished studies in healthy adults and four unpublished toxicity studies in rats (acute oral toxicity, 30-day repeated dose oral toxicity, and two 6-month repeated dose oral toxicity studies) and its history of consumption in the United States and worldwide. The notifier notes that in 1998, the FDA filed without comment a New Dietary Ingredient notification for the use of L. casei in dietary supplements. The notifier also notes that in 2007, the European Food Safety Authority placed both L. casei and L. paracasei on its list of bacteria eligible for Qualified Presumption of Safety and that this opinion was restated in 2011 supporting its safe use. Based on all the data and information available, the notifier concludes that L. casei strain Shirota is GRAS under the conditions of its intended use.

Standards of Identity

In the notice, Yakult states its intention to use L. casei strain Shirota in fermented dairy products, including foods for which standards of identity exist, located in Title 21 of the Code of Federal Regulations. We note that an ingredient that is lawfully added to food products may be used in a standardized food only if it is permitted by the applicable standard of identity.

Potential Labeling Issues

In describing data and information on clinical studies that Yakult relies on to conclude that L. casei strain Shirota is GRAS under the conditions of its intended use, Yakult raises a potential issue under the labeling provisions of the Federal Food, Drug, and Cosmetic Act (FD&C Act). Under section 403(a) of the FD&C Act, a food is misbranded if its labeling is false or misleading in any particular. Section 403(r) of the FD&C Act lays out the statutory framework for the use of labeling claims that characterize the level of a nutrient in a food or that characterize the relationship of a nutrient to a disease or health-related condition. If products that contain L. casei strain Shirota bear any claims on the label or in labeling, such claims are the purview of ONLDS in CFSAN. The Office of Food Additive Safety (OFAS) neither consulted with ONLDS on this labeling issue nor evaluated the information in your notice to determine whether it would support any claims made about L. casei strain Shirota on the label or in labeling.

Allergen Labeling

The Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA) amends the Federal Food, Drug, and Cosmetic Act to require that the label of a food that is or contains an ingredient that bears or contains a “major food allergen” declare the presence of the allergen (section 403(w)). FALCPA defines a “major food allergen” as one of eight foods or food groups (i.e., milk, eggs, fish, Crustacean shellfish, tree nuts, peanuts, wheat, and soybeans) or a food ingredient that contains protein derived from one of those foods. L. casei strain Shirota grown in a milk-based media may require labeling under FALCPA, because it may contain protein derived from milk. Although issues associated with labeling food are the responsibility of ONLDS, issues associated with FALCPA exemptions are the responsibility of the Food Allergen Coordinator in OFAS.

Section 301(ll) of the FD&C Act

The Food and Drug Administration Amendments Act of 2007, which was signed into law on September 27, 2007, amends the FD&C Act to, among other things, add section 301(ll). Section 301(ll) of the FD&C Act prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FD&C Act, a biological product licensed under section 351 of the Public Health Service Act, or a drug or a biological product for which substantial clinical investigations have been instituted and their existence made public, unless one of the exemptions in section 301(ll)(1)-(4) applies. In its review of Yakult’s notice that L. casei strain Shirota is GRAS for the intended use, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing L. casei strain Shirota. Accordingly, this response should not be construed to be a statement that foods that contain L. casei strain Shirota, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).


Based on the data and information provided by Yakult, as well as other information available to FDA, the agency has no questions at this time regarding Yakult’s conclusion that L. casei strain Shirota is GRAS under the intended conditions of use. The agency has not, however, made its own determination regarding the GRAS status of the subject use of L. casei strain Shirota. As always, it is the continuing responsibility of Yakult to ensure that food ingredients that the firm markets are safe, and are otherwise in compliance with all applicable legal and regulatory requirements.

In accordance with proposed 21 CFR 170.36(f), a copy of the text of this letter responding to GRN 000429, as well as a copy of the information in this notice that conforms to the information in the GRAS exemption claim (proposed 21 CFR 170.36(c)(1)), is available for public review and copying at


Dennis M. Keefe, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition

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