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BIOACTIVE FOODS IN PROMOTING HEALTH: PROBIOTICS AND PREBIOTICS
Academic Press
Copyright © 2010 Elsevier Inc.
All right reserved.
ISBN: 978-0-08-095854-5
Chapter One
Production of Probiotic Cultures and Their Incorporation into Foods Edward R. Farnworth and Claude Champagne Food Research and Development Centre, Agriculture and Agri-Food Canada Saint-Hyacinthe, Quebec, Canada
1. INTRODUCTION
Our understanding of the population of bacteria that inhabit the gastrointestinal tract is increasing, and it is becoming more evident that the makeup of this large diverse bacterial community impacts on our digestion, metabolism and health. The chapters in this book illustrate the many and varied ways in which human health might be improved by the consumption of live bacteria.
Many experiments involving the feeding of probiotic bacteria have used the organism of interest either alone, or added to milk or yogurt. However, as the number of bacteria identified with beneficial properties has grown, there has been increasing interest in expanding the type of foods into which these beneficial bacteria could be added. However, live bacteria often have strict nutrient requirements for growth, and their viability can be dependent on the environment (food matrix) in which they are located.
A commonly accepted definition of probiotics is that they are 'live microorganisms which, when administered in adequate amounts, confer a health benefit on the host'. This implies that cells must be alive when consumed, and explains why the focus of this chapter is on the delivery of viable cells. However, there are instances where a beneficial effect derived from a probiotic culture does not need live cells. We shall address this aspect and introduce the concept of 'probioactives.'
Furthermore, in the definition of probiotics given above, the efficacy of probiotic-carrying foods can only be assured when: a) beneficial bacteria have been added to foods and beverages in sufficient numbers; b) means have been found to minimize harmful/food matrix interaction; and c) viability has been maintained during manufacture, storage and consumption. This chapter will detail the challenges that face food manufacturers who wish to add bacteria to their probiotic products, and outline some of the solutions that have allowed for the development of an ever-growing diverse line of probiotic foods.
2. PRODUCTION OF PROBIOTIC CULTURES FOR FOODS OR FOOD SUPPLEMENTS
The technology related to the production of probiotic cultures by specialized suppliers has already been reviewed. Therefore, this section will not focus on the production parameters that affect the biomass yields. Rather, emphasis is placed on production parameters as they pertain to subsequent viability of the cultures in stressful conditions. The more cells are able to survive stressful conditions in food processing and storage, and/or the stomach, the greater is their viability once they reach to the intestines.
A summary of the production parameters that impact on the ability of probiotic bacteria to survive challenges in the food and the gastrointestinal tract (GIT) are presented in Table 1.1. The first parameter, strain selection, is arguably the most important. Lactic cultures are notorious for variability (even within a given species) of their abilities to grow on food matrices as well as survive heating, freezing, or storage in acid environments. In the past, probiotic cultures destined for addition to foods were chosen, therefore, mainly on their technological properties. However, the requirement for demonstrated health effects has resulted in this parameter increasingly being the principal element of strain selection for food applications. In this situation, production and food processing parameters must be adapted to prevent lethal or sub-lethal damages to cells.
Production parameters of probiotic cultures can be adapted at the fermentation, concentration, stabilization, or storage levels (Table 1.1). For example, fermentation temperature modifies the composition of bacterial membranes. A first concept that must be emphasized is that cells can have sub-lethal damage due to processing parameters. This damage can be to the cell wall or the membranes. Presumably, denaturation of internal cell components (for example enzymes) would also generate such sub-lethal damage. It is easy to visualize how high pressures, freezing, and drying can have such effects on cells, and how they result in cultures having lower subsequent resistance to detrimental environmental conditions. But it is less obvious how the fermentation conditions can damage the cells. For example, with lactic cultures, it is well known that extensive over-incubation of a starter with or without pH control, will result in lower specific acidifying activity. A second concept that warrants mention is that applying limited controlled stresses can actually increase the ability of cultures to survive subsequent harsh conditions. As an example, when Lactobacillus delbrueckii ssp. bulgaricus cells were submitted to a heat pre-treatment at 50°C or to a hyper-osmotic pre-treatment, the viability of cells to a lethal temperature challenge (65°C) increased. A small heat pre-treatment can also improve survival to freeze-drying. Other data show how sub-lethal acid shocks improve viability to heating or freezing. From these two concepts it is clear that biomass production parameters modify the resulting cells; sometimes to their disadvantage, sometimes to their benefit. This requires research and a stringent process control to successfully produce probiotic cultures with an improved ability to be delivered in a viable state in foods following harsh processing conditions.
3. ENSURING DELIVERY OF VIABLE CULTURES IN FOODS AND SUPPLEMENTS
3.1. Delivering as Food Supplements
Supplements are typically delivered in caplets or capsules. The ability of the products to deliver probiotics is mainly set at the production level and, for consumers, storage then becomes the main issue for viability. There are three principal factors which influence the viability of probiotics during storage: temperature, oxygen and relative humidity.
As a rule, cultures, even dried, should be kept refrigerated. In traditional freeze-drying processes, increasing the storage temperature from 4 to 25°C results in a ten-fold reduction of stability. Some commercial products can be kept at room temperature over a few months and do not suffer losses in viability greater than 1 log. However, highly specific and controlled manufacturing conditions are required to obtain such products. As a result, there are reports of products, often inappropriately stored on the shelves, that do not have the claimed populations. Another problem is the fact that strains do not die at the same rate during storage. Thus, the 'total' population in the product might be correct, but the strain ratios could be significantly modified during storage.
Moisture is the second parameter to consider. As a rule, dried cultures should have a water activity (aw) content of 0.1, and high losses in viability occur above an aw of 0.3. During storage it is imperative that the moisture in the air be able to increase the aw of the culture powder. To prevent exposure of the cultures to water during storage, two actions are taken by companies: 1) packaging in water-impermeable bottles or films; and 2) addition of small moisture-binding sachets in bottles. These strategies work well until the packaging is opened. From this point on, the stability of the culture will depend on the amount of water that is absorbed by the product, especially when the packaging bottle is repeatedly opened.
Finally, oxygen is detrimental to the viability of probiotics during storage. To enhance stability during storage, companies typically add antioxidants in the drying medium. Oxygen binders also exist in sachets, but they are not used nearly as much as the water-absorbing ones. As for moisture, this protection is reduced when the product is opened.
All of these elements point to desirable practices for consumers who wish to receive the maximum delivery of probiotics through caplet or capsule supplements:
1. Keep products refrigerated, even if the label states that the cultures are stable at room temperature.
2. Close the bottle as rapidly as possible once the supplement is taken, in order to reduce the entrance of oxygen and moisture into the bottle.
3. If water-absorbing or oxygen-binding sachets are present in the bottle, do not remove them.
3.2. Delivering by Processed Foods
The first foods with probiotic bacteria were yogurts, and fermented milks are still the most important food vehicle for the delivery of probiotic bacteria. However, other foods have now appeared which carry probiotic bacteria. Numerous entries in the functional food market are linked to beverages, such as unfermented milk and fruit juices. Cheese is also gaining acceptance in the market. In addition to these commercial products, many research projects have been carried out which propose the addition of probiotics to chocolate, sausages, cereal products, dried products and vegetables. A multitude of food products contain lactic cultures and are subject to enrichment by probiotic bacteria. Therefore, the potential of delivery of probiotic bacteria by foods is immense.
The first question, with respect to delivering probiotics in foods, is 'how do we add the cultures to the food matrix?' With the exception of very large companies, probiotic cultures are not prepared at the food processing plant but, rather, added directly to the vat. This is sometimes called 'direct to the vat inoculation' (DVI). Various reasons explain this, but mostly it is for greater flexibility, and to better standardize the delivery of the cultures. DVI can be carried out by simply opening the sealed packaging and adding the frozen or dried culture to the food matrix. Although it appears easy, if done inappropriately it can lead to substantial losses in viability. Indeed, how a culture is thawed or hydrated can result in a ten-fold variation in colony-forming units (CFU). With respect to frozen cultures, the thawing temperature needs to be selected, but few other thawing parameters seem to require specific adjustments. This makes inoculation with frozen cultures rather easy, and few mistakes can be made. This is not the case with the freeze-dried cultures. Although dried cultures are much easier to ship and store than frozen ones, their use in the food processing plant is more difficult. In addition to the plating medium itself, four rehydration parameters influence CFU counts following addition of a powder in a food matrix (Table 1.2). It should be mentioned that these data could also be applied to clinicians wishing to provide probiotics to patients through foods. Rehydration of a powder into a cold fruit juice and drunk immediately, for example, introduces three conditions (low temperature, high acidity, no recovery period) which potentially generates viability losses. A question thus arises: 'could probiotic preparation techniques be responsible for wide variations of inoculation level and, hence, variable clinical effects?'
The second point to consider is 'can the probiotic cultures survive the processing steps?' Processing of foods requires various technological steps, and many are detrimental to the viability of probiotic bacteria. Examples are presented in Table 1.3. It can be seen that viability losses sometimes reach 6 logs. Reviews of the challenges which occur during food processing have been published, and the reader is referred to these publications for examples of applications. To prevent viability losses during processing, two main strategies have proven successful:
1. Modify the food matrix
a. pH (neutral pH preferable)
b. addition of antioxidants
c. addition of growth factors (prebiotics, plant or yeast extracts)
d. selection of non-toxic ingredients (flavours, preservatives).
2. Modify the process
a. lower temperatures
b. include vacuum or nitrogen flushing
c. modify the fermentation parameters (selection of compatible starter culture, inoculation rate, enzymes)
d. adapt cells by applying sub-lethal stresses (thermal, pH, osmotic).
Although adapting media and processing conditions may seem easy, it is not. As an example, in the development of a new fermented milk containing probiotic bacteria, 21 parameters can be considered (Table 1.4).
A third point to consider is storage. Unfortunately, processing parameters are not the only elements which affect the delivery of viable cells to consumers in foods. As was the case for supplements, viability losses occur during storage. Again temperature, moisture and oxygen constitute factors which affect the extent of population losses. However, in foods, additional factors must be mentioned: nature of the starter culture, pH, redox level, type of packaging. Storage also not only affects the viability of cells per se, but also the ability of the viable cells to survive the harsh environment of the GIT following consumption. Thus, cultures of lactobacilli were much more sensitive to low pH similar to that in the stomach, when they had been stored for 35 days in a fruit juice blend (Table 1.5). Fortunately, the ability to survive exposure to bile salts was not affected by this 35-day storage period in the juice ( Table 1.5 ). Little is known on how storage can affect the subsequent functionality of probiotic bacteria, and more research is needed in this area.
Finally, the question of how viability can be affected at consumers' homes has received little attention. With beverages in large containers (greater than 1 L), bottles are opened, a portion is taken (typically 250 mL), and the remainder is replaced in the refrigerator. Since some probiotic bacteria are quite sensitive to oxygen, a concern can be raised on the detrimental effect of oxygen on the cells found in the remaining beverage. With L. rhamnosus R0011 this has not been found to be a problem, but studies on other cultures, particularly bifidobacteria, seem warranted.
4. ADDITION OF PROBIOTICS TO FOODS—ENSURING EFFICACY
4.1. Effective Dose
Consumers who are looking to add probiotic bacteria to their diet have several questions to ask themselves. The first question is—'which bacteria to consume? ' The science behind the beneficial effects of consuming probiotic bacteria is expanding. Although there have been a large number of diseases/health conditions that have been the target of probiotic treatment studies, a consensus on the effectiveness of probiotics for specific uses in humans is limited to a few applications at the present time. This reality is emphasized by the fact that, to date, very few probiotic products have received health claims approved by health regulatory bodies.
The second and equally important question is that of dose and duration of the consumption. Because of the lack of clear scientific evidence to show the level of consumption to ensure efficacy, the industrial strategy appears to have been to add as many live bacteria to a food product as is technically and economically realistic. This inevitably results in the conclusion on the part of the consumer that 'more is better.' It is difficult to find published data for proposed probiotic bacteria to satisfy the major part of the probiotic definition—'... administered in adequate amounts confer a health benefit ...' —a definition that clearly requires demonstration of the effective dose.
It has to be emphasized that the minimum number will vary depending on the bacteria (at the species and sub-species level) being used, the form in which it is consumed (as part of a food or in a capsule or pill), and the application it is being used for. The scientific literature contains a large range for the number of bacteria that have been suggested to produce a probiotic effect; 105 colony-forming units (CFU) as a 'therapeutic minimum' to 1011. Unlike studies of new drugs, dose response studies for probiotic bacteria are not common.
The Fermented Milks and Lactic Acid Bacteria Beverages Association in Japan have set a minimum of 107 bifidobacteria/g or mL for fermented milk products in Japan. CODEX has set a minimum of 106 CFU /g for microorganisms added (in addition to those added to produce the product) to fermented milk and yogurt. Recommendations for foods other than fermented milk are not evident at this time.
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Excerpted from BIOACTIVE FOODS IN PROMOTING HEALTH: PROBIOTICS AND PREBIOTICS Copyright © 2010 by Elsevier Inc.. Excerpted by permission of Academic Press. All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
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