Elsevier

Food Research International

Volume 83, May 2016, Pages 95-101
Food Research International

Lactobacillus casei LcY decreases milk protein immunoreactivity of fermented buttermilk but also contains IgE-reactive proteins

https://doi.org/10.1016/j.foodres.2016.02.016Get rights and content

Highlights

  • Fermentation with L. casei LcY decreased immunoreactivity of sweet buttermilk proteins.

  • Digestion further reduced the immunoreactivity of fermented buttermilk proteins.

  • L. casei LcY contains proteins which react with food-allergy patients' serum.

  • Lactic acid bacteria's allergenic potential requires further study.

Abstract

This study evaluates Lactobacillus casei LcY protein immunactive properties and assesses its potential to decrease milk protein immunoreactivity in fermented buttermilk before and after simulated digestion. Competitive ELISA analysis of α-lactalbumin, β-lactoglobulin, α-casein, β-casein, κ-casein, bovine serum albumin and lactoferrin determined significant (P < 0.001) immunoreactivity reduction after fermentation, and even greater decrease after three-stage simulated digestion. Immunoblotting of fermented buttermilk with human allergic sera revealed that α-casein remained the most allergenic milk protein. The LcY cell fractionation and further separation by SDS-PAGE and 2DE, immunoblotting and MALDI-TOF MS/MS identification all highlighted that cyclopropane-fatty-acyl-phospholipid synthase and carboxylate-amine ligase in the cell wall/membrane protein fraction reacted with human IgE antibodies. In silico analysis of these proteins' allergenic potential indicated that these are new allergens rather than already known cross-reacting allergens.

To our best knowledge, this is the first study providing evidence of human IgE reactive protein presence in lactic acid bacteria.

Introduction

The World Allergy Organization statistics highlight that 11–26 million people in the European Union and 220–520 million world-wide suffer from food allergies (Pawankar, Holgate, Canonica, & Lockey, 2011). However, no specific foods are currently blamed for human gastrointestinal tract symptoms, including food allergy. Therefore, ongoing scientific effort seeks to create a low antigenic diet which counteracts food allergy. Some neonate and child studies suggest that fermented milk products with reduced immunoreactivity and probiotic bacteria aid allergy prevention and treatment (Giovannini et al., 2007, Morisset, M., et al., 2011), and lactic acid bacterial beneficial effects have been examined in several clinical studies on atopic eczema and dermatitis. However, reports on Lactobacillus rhamnosus GG effect vary. Kalliomaki et al. (2001) and Kalliomaki, Salminen, Poussa, Arvilommi, and Isolauri (2003) probed that L. rhamnosus GG could reduce atopic eczema in infants born to women taking prophylactic probiotic preparation containing the mentioned strain, but other studies showed no significant difference between patients administered verum or a placebo (Kukkonen et al., 2007). In addition, Grüber et al. (2007) recorded no L. rhamnosus GG therapeutic effect in mild-to-moderate atopic dermatitis, and Taylor, Dunstan, and Prescott (2007) observed increased allergen sensitization after L. acidophilus administration. Although the varied probiotic effects on immune reaction are explained by strain heterogeneity, dosage and therapy duration (Castellazzi et al., 2013), no scientific data currently exists on lactic acid bacteria (LAB) protein influence on allergic patients' immune systems.

In addition to defined probiotic strains, many other Lactobacillus strains are applied in the food industry because of their technological benefits. For example, Lactobacillus strains are used either as starter cultures or additives in the production of fermented foods including yogurt, meats and sausages (De Vuyst, L., et al., 2008, Leroy, F. and De Vuyst, L., 2004, Rubio, R., et al., 2014). Fermented milk beverages are generally produced from pasteurized, homogenized and normalized fat-content milk and isolated milk proteins are added to provide the protein-rich environment required for successful bacterial growth. LAB proteolytic activity is vital for their growth in the food matrix, but the extent of this activity differs significantly in different strains (Pescuma, Hébert, Bru, Font, & Mozzi, 2012). Moreover, although bacterial enzymatic proteolysis provides essential components which improve product sensory qualities, it can also alter milk protein immunoreactivity. Previous studies on fermented milk products indicate the application of selected LAB strains (Jędrychowski & Wróblewska, 1999) and also the subsequent enzymatic and chemical modification of technological processes in reducing milk protein allergenicity (Wróblewska, B. and Jędrychowski, L., 2002, Jędrychowski, L., et al., 2005, Wróblewska, B., et al., 2013).

Buttermilk is a valuable by-product obtained in butter production and possesses nutritional properties similar to milk. Most of the buttermilk as a by-product is a burden on the environment, although some is utilized in fertilizers and also in bread production (Bilgin, Daglioglu, & Konyali, 2006). Herein, we utilize buttermilk from butter production as the Lactobacillus growth substrate and we examine the effect of sweet buttermilk fermentation with Lactobacillus casei LcY on immunoreactivity and allergenicity of the fermented beverage proteins. Special emphasis is placed on the allergenic potential of extracellular and intracellular proteins of this bacterial strain.

Section snippets

A strain and culture conditions

A strain representing one of the most common LAB species (L. casei) used in the food industry was selected based on results of preliminary studies concerning the potential of LAB to reduce immunoreactivity of milk proteins. The tested L. casei LcY was obtained from the Collection of Microorganisms of the Microbiology Laboratory at the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences (Olsztyn, Poland). The strain isolated from a fermented milk beverage had

Fermented buttermilk immunoreactivity and allergenicity

Fermentation reduced the immunoreactivity of the main milk protein allergens examined with specific rabbit polyclonal antibodies (Table 1A). The highest reduction was determined for α-la and β-lg by up to 97 and 95%, respectively. The level of immunoreactivity of α-cas, a major milk allergen, also decreased during fermentation to 21%. Although BSA and lactoferrin are not considered strong allergens, their immunoreactivity was also lowered by bacterial activity to 65 and 89% of the unfermented

Discussion

While many studies have focused on analysis of the proteolytic activity of lactic acid bacteria used in fermented beverages and on the immunoreactivity of products such as yogurt, whey, curds and kefir (Bu, G., et al., 2010, Jędrychowski, L. and Wróblewska, B., 1999, Kabadjova-Hristova, P., et al., 2006, Law, B.A., 1997), no known reports exist on the influence of fermentation on the allergenic potential of beverages obtained from sweet buttermilk. In this study we document L. casei LcY's

Conclusions

The results of this study showed that sweet buttermilk fermentation with L. casei LcY significantly lowered the immunoreactivity and allergenicity of main whey milk proteins. The simulated three-stage digestion of fermented buttermilk further reduced the immunoreactivity of the analyzed allergens. We have confirmed that cyclopropane-fatty-acyl-phospholipid synthase and carboxylate-amine ligase in the L. casei LcY cell wall/membrane fraction react with human IgE antibodies.

Acknowledgments

This study was funded by the European Union Social Fund (DrINNO3), by the Ministry of Science and Higher Education's Project No. N N312 311939 and by the statutory fund of the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences. The authors thank Ewa Romaszko MBBS and Magdalena Zakrzewska MD for providing human serum and also the Regional Dairy Cooperative in Błonie, Poland for providing sweet buttermilk.

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