Salts and sugars
Salt is probably the most common microbial inhibitor in food products and is a popular constituent of seasoning blends and concentrated flavour bases. When used at optimal concentration in aqueous solutions (0.85-0.90 percent), salt maintains an isotonic solution where water passes equally from the microorganism to the outer environment and vice versa. However, at concentrations >5 percent, water passes out of the cell at a greater rate than it enters in a phenomenon called plasmolysis.1 This results in dehydration of the cell, which can lead to microbial growth inhibition or even death.
Sugar (such as sucrose, lactose and fructose) acts in the same manner, but requires a much higher concentration to achieve the same effect (ie, it takes six times more sucrose than salt to achieve the same level of microbial inhibition).1 In general, an increased enrichment dilution, like 1:20, may be sufficient to overcome the inhibitory effects of salts and/or sugars, but the dilution can increase up to 1:200 depending on concentration or if other inhibitory compounds are present.
Thickening agents
Food matrices that possess thickening properties are not inherently inhibitory but may entrap target bacterial cells and isolate them from the enrichment media. For starchy products (cereals, snack puffs, cookies, potato flakes, oats, fibre, pure starch), an enrichment in buffered peptone water (BPW) supplemented with α-amylase can be utilised at 1:10, in most cases. Higher enrichment dilutions up to 1:100 may be required however, depending on the strength of the thickening properties. Gums (xanthan, carrageenan, guar, etc) may need at least a 1:50 enrichment dilution without supplementation, or supplementation with one percent cellulase to reduce viscosity.2
Figure 1: Example protocol of a quantitative matrix verification
Egg powders can vary in terms of thickening properties, with some products requiring a 1:10 enrichment, while other varieties may need up to 1:100.
Oils, solid fat, or products containing high amounts of fat
This category of products is not microbially inhibitory, but microorganisms may be present in the fat layer, which can prevent them from contacting the aqueous enrichment broth. Examples of these products can include oils, solid fat (eg, tallow), cheeses, sauces (pesto or alfredo), coffee creamers, dressings, flavour bases, coconut, legumes, seeds, nut butters, lecithin and cooked (fried) or cured meats. To aid solubility of the fat layer with the enrichment media, BPW supplemented with one percent Tween 80 and an appropriate enrichment dilution can be used.
Flavouring agents and extracts
Liquid flavourings can pose a challenge for the microbiology laboratory due to the proprietary formulations of the product, as well as their ingredients. These products can contain ethanol that serves as a carrier solvent and flavour enhancer. However, ethanol is well known for its lethal desiccant and denaturant effects on microorganisms.1 Another component of liquid flavours is propylene glycol, which is generally recognised as safe (GRAS) and is used to evenly distribute the flavour and maintain flavour quality.5 Propylene glycol is also known to be inhibitory to various microorganisms.6
Vanilla, specifically its compound vanillin, is another common flavouring agent that is known to be inhibitory or even lethal to microorganisms by negatively affecting enzymatic processes, membrane integrity, or genetic material.1,7 Flavourings can vary in microbial inhibition depending on their composition; therefore, it is essential to determine the appropriate enrichment dilution. At NQAC Dublin, we have found that enrichment dilutions can go as high as 1:3000, especially if vanilla or cinnamon are part of the flavouring components.
Herbs and spices
Herbs and spices constitute a large group of plant products with numerous food applications – as additives, flavourings, and even as antimicrobials/preservatives.3,4,8 Food items in this group that NQAC Dublin commonly encounters include onion, garlic, basil, thyme, oregano, clove, cinnamon, rosemary, sage, coriander, paprika, turmeric and ginger. Each herb or spice has its own unique constituents that provide antimicrobial characteristics, including phenolic compounds, essential oils, organic acids, aldehydes, ketones, alcohols, or sulphur-containing compounds, with essential oils and phenolics being the most potent.2
Figure 2: Example protocol of a qualitative matrix verification
Onions and garlic are a common constituent of products received for testing, and they can be submitted in a minced/minimally processed form, processed (ie, dehydrated, powdered, roasted), or as an ingredient that is part of a seasoning blend/flavour base/sauce/finished product. To overcome the inhibitory effect of onions and garlic, enrichment in BPW supplemented with 0.5 percent of potassium sulphite (K2SO3) can be used, but an appropriate dilution must still be determined (which can range from 1:10 to 1:100). Based on our experiences, herbs and spices such as turmeric, sage, paprika, ginger and black pepper tend to be the least inhibitory (enrichment at 1:10, maybe 1:20) while basil, garlic, oregano, coriander, cinnamon, rosemary and cloves tend to be the most inhibitory (enrichment dilutions up to 1:1000 depending on the herb/spice).
Background flora/probiotics
Food products with a naturally high background flora can potentially interfere with detection or quantification of target organisms. High levels of background flora can provide competition for nutrients, cause unfavourable changes to the surrounding environment (ie, production of acids), or produce antimicrobial substances (ie, bacteriocins like nisin) that can inhibit or inactivate target organism cells.1
Probiotic products can produce the same effect. Probiotic formulations may include multiple strains and colony-forming unit (CFU) levels in the millions or billions per dose. When analysing products with high background flora or probiotics, different strategies can be employed to suppress the background/probiotic organisms or provide more opportunities for the target organism to grow. Aside from increasing the initial enrichment dilution, other strategies can include supplementation of the enrichment broth with an antibiotic (ie, vancomycin), using enrichment media with increased buffering capacity (ie, double or 6-strength BPW), or a combination of these strategies.
Matrix verifications
If the appropriate laboratory facilities are available, food matrix verification may be done in-house or can be performed by a commercial laboratory. Figures 1 and 2 give examples of how qualitative and quantitative matrix verifications are performed, respectively. In general, this involves spiking food matrices with the target organism, preparing various enrichment dilutions using the appropriate media, and performing analysis using the intended analytical platform. Based on the results, the correct enrichment dilution and enrichment media will be determined for use for future samples. Additionally, including a list of potentially inhibitory ingredients to the laboratory can help expedite the matrix verification process.
Conclusion
Knowledge of the potential inhibitory substances or challenging properties that exist in a food item aids in proper detection or quantification of the target organism. Matrix verifications are an essential tool for the food microbiology laboratory, and active collaboration with the customer ensures timely and accurate results.
About the author
Dr Gabriel C Sanglay is a Senior Microbiologist at the Nestle Quality Assurance Center (NQAC) in Dublin, Ohio and has been a member of the Biochemistry & Special Investigations department for more than seven years. His areas of expertise include method validation and verification, new method implementation, cultural and molecular detection of pathogens, food matrix verifications, and probiotics. Prior to NQAC Dublin, Gabe obtained his degrees in Food Science & Technology at Virginia Polytechnic Institute and State University (BS and MS), and The Ohio State University (PhD). To date, he has authored/co-authored 12 peer‑reviewed publications during his career. Gabe has previous experience as a Microbiologist for the USDA Agricultural Research Service in Beltsville, Maryland, where he worked for five years, and over 10 years of experience in the academic sector serving as a graduate research associate and as a USDA‑NIFA postdoctoral fellow.
References
- Jay JM, Loesser MJ, Golden DA. Modern Food Microbiology, 7th ed. Chapter 13 – Food Protection with Chemicals and by Biocontrol. 2005.
- Gurtler JB, Keller SE, Kornacki JL, et al. 2019. Challenges in recovering foodborne pathogens from low-water activity foods. J. Food Prot. 82(6):988-996.
- Quinto EJ, Caro I, Villalobos-Delgado LH, et al. 2019. Food safety through natural antimicrobials. Antibiotics. 8, 208. https://doi.org/10.3390/antibiotics8040208
- Lucera A, Costa C, Conte A, Del Nobile MA. 2012. Food applications of natural antimicrobial compounds. Front. Microbiol. 3, 287. https://doi.org/10.3389/fmicb.2012.00287
- Food Insight. 2014. “Questions and Answers about Propylene Glycol”. https://foodinsight.org/questions-and-answers-about-propylene-glycol/
- Nalawade T, Bhat K, Sogi S. 2015. Bactericidal activity of propylene glycol, glycerine, polyethylene glycol 400, and polyethylene glycol 1000 against selected microorganisms. J. Int. Soc. Prev. Community Dent. 5(2): 114-119.
- Krushnamurthy A, Shyamala B, Naidu M. 2013. Vanilla – its science of cultivation, curing, chemistry, and nutraceutical properties. Crit. Rev. Food Sci. Nutr. 53:12, 1250-1276.
- Liu Q, Meng X, Li Y, et al. 2017. Antibacterial and antifungal activities of spices. Int. J. Mol. Sci. 18, 1283. https://doi.org/10.3390/ijms18061283
