A feasibility study of Lactobacillus plantarum in ... - Wiley Online Library

International Journal of Food Science and Technology 2016, 51, 381–388

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Original article A feasibility study of Lactobacillus plantarum in fruit powders after processing and storage Sandra Borges, Joana Barbosa, Joana Silva, Ana M. Gomes, Manuela Pintado, Cristina L. M. Silva, Alcina M. M. B. Morais & Paula Teixeira* CBQF-Centro de Biotecnologia e Quımica Fina – Laborat orio Associado, Escola Superior de Biotecnologia, Universidade Cat olica Portuguesa/Porto, Rua Arquiteto Lob~ao Vital, Apartado 2511, Porto 4202-401, Portugal (Received 22 May 2015; Accepted in revised form 9 September 2015)

Summary

The aim of this study was to develop fruit powders (apple, banana and strawberry) enriched with a probiotic strain (Lactobacillus plantarum 299v). Two methodologies were proposed: (i) drying of the fruit with probiotic culture incorporated (by convection) or (ii) drying of fruit (by convection) and addition of spray-dried probiotic culture. In the first methodology, processing caused a notable reduction in probiotic viable counts in apple, but this reduction was lower during drying of banana and strawberry. A large reduction in viable cells was also recorded during storage. In the second methodology, the survival of L. plantarum 299v was considerably higher during spray-drying, and fruit powders with a microbial content suitable for a probiotic food (108–109 cfu g1) were obtained. The fruit powders incorporating L. plantarum 299v can be stored at 4 °C or at room temperature, for at least 3 months. This preliminary study demonstrated that fruit powders are good carriers of probiotic cultures, but the techniques used to produce them should be carefully considered.

Keywords

Drying by convection, probiotic fruit powders, probiotic survival during storage, spray-drying.

Introduction

Currently, a balanced diet is regarded as a vital key to continued good health. Thus, the development of food products that promote health is a priority for the food industry. This general trend has been seen in the consumption of products with physiologically active compounds, such as probiotics (Mitropoulou et al., 2013). It is estimated that the probiotics industry holds 10% of the market share of the global functional food market, probiotic products becoming a substantial and increasing sector of economic importance (Champagne et al., 2011). Most microbial strains used as food and feed probiotic supplements belong to Lactobacillus and Bifidobacterium genera as a result of their recognition as part of the indigenous microbiota of humans and animals, their safe use and evidence that indicates they play important roles in health promotion (Gaggia et al., 2011). Several studies have described the health benefits of the ingestion of probiotics including, for example, prevention and treatment of gastrointestinal diseases (Gagnon et al., 2006; Ritchie & Romanuk, 2012; del Campo et al., 2014), anti-allergic effects (Lee *Correspondent: E-mail: [email protected]

doi:10.1111/ijfs.12975 © 2015 Institute of Food Science and Technology

et al., 2014), anticarcinogenic properties (Kumar et al., 2012) and reduction in cholesterol (Wang et al., 2012; Miremadi et al., 2014). Although there is not a generally recognised number of viable cells consumed to ensure a health effect, recommendations are provided by various agencies, such as the Food and Agriculture Organization/World Health Organization (2002) and the Canadian Food Inspection Agency (2009). A product that includes probiotic micro-organisms should contain an amount of viable cells at the point of consumption that have been shown to be efficacious, which is usually >106–108 cfu g1 or >108–1010 cfu day1 (Champagne et al., 2011). Probiotics are generally added to fermented dairy products such as yogurt, cheese, ice cream and butter. As an interim step in this development, hybrid dairy products have been introduced in the market, combining dairy products and fruit beverages to provide more convenient and more variety of flavoured products (Khurana & Kanawjia, 2007). Fruits are an important source of energy, vitamins, antioxidants, minerals and dietary fibre. This naturally nutritious composition of fruits confers health protective benefits, preventing free radicals from damaging proteins, DNA and lipids (Orrego et al., 2014). Therefore, the consumption of fruits could reduce the risk of

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Lactobacillus plantarum in fruit powders S. Borges et al.

ischaemic heart disease, ischaemic stroke, stomach, lung and colorectal cancer (Lock et al., 2005). One of the barriers to fruit consumption is the time required to prepare them. Moreover, many fruits deteriorate rapidly during and after preparation, for example enzymic and/or nonenzymic browning, so the preparation should be suitable to maintain the nutritional value at the time of consumption and during the storage. Consumers want products that are available throughout the year, suitable for many uses and with an extended shelf life (Orrego et al., 2014). Dried fruits have several advantages such as lower storage costs and the stabilization of active substances (Krishnaiah et al., 2014). Drying techniques are also used to produce dehydrated probiotic cultures to stabilize the culture and facilitate the storage, handling, transport and their use in functional foods (Meng et al., 2008). It is essential to obtain and maintain high cell viability during drying and storage; therefore, these steps present particular challenges in the production of commercial probiotics. The development of suitable methods to maintain the viability and efficacy of the probiotic population is extremely important to the health food industries (Gaggia et al., 2011). The food matrices have different physico-chemical properties that may affect the viability of the probiotic strain, for example the behaviour of the probiotic strain in a liquid matrix (e.g. yogurt) is different from a solid matrix (e.g. fruit). So, the interaction of the probiotic and food matrices must also be considered when developing functional foods. Thus, the aim of this study was to develop fruit powders (apple, banana and strawberry) incorporating a probiotic strain, using drying technologies, and to evaluate the viability of the bacteria in these fruit matrices during processing and storage. Material and methods

Probiotic strain and growth conditions

Lactobacillus plantarum 299v (Probis Probiotika, Lund, Sweden) was the commercial probiotic strain selected for this study. Besides the health benefits, this strain does not possess virulence traits nor significant antibiotic resistances (Barbosa et al., 2014). The probiotic strain was grown on de Man, Rogosa and Sharpe (MRS) agar (Lab M, Bury, UK) and was subcultured twice in MRS broth (Lab M) at 37 °C for 24 h before use in all tests. Stock cultures were maintained in MRS broth supplemented with 30% (v/v) of glycerol (Panreac, Barcelona, Spain) and stored at 80 °C. Fruit

Fruit cubes (1.0 cm) of apple (Granny Smith), strawberry (mixture of Festival, Camarosa and other local

International Journal of Food Science and Technology 2016

varieties) and banana (Cavendish) were gratefully provided by FrulactÒ. Drying of fruit with probiotic

To develop the fruit powder incorporating the probiotic, two different experimental procedures were followed. In the first experiment, the fresh fruit was incorporated with L. plantarum 299v by immersion and then dried. In the second experiment, the fruit and the probiotic strain were dehydrated separately and then, the dried probiotic culture was added to the dried fruit. The assays were performed as described below, and they are shown schematically in Fig. 1. Drying of the fruit with probiotic culture incorporated (by convection) Sample preparation

The probiotic culture L. plantarum 299v was grown as previously described (obtaining a cell density of approximately 1010 cfu mL1), centrifuged (8877 g at 4 °C; Rotina 35R, Hettich, Germany) for 5 min and deposited cells washed twice by centrifugation in sterile Ringer’s solution (Oxoid, Hampshire, UK). The harvested cells were re-suspended to the original volume in Ringer’s solution or in reconstituted skim milk powder (11% w/v; Oxoid); the inoculation level of the probiotic was ca. 1010 cfu mL1 in each case. The fresh fruit pieces (apple, strawberry or banana) were immersed in these probiotic suspensions (in Ringer’s and skim milk) for 1 h, in order to promote the adherence of the bacterial cells to the fruit. The following parameters were maintained: the initial level of inoculum in Ringer’s solution and skim milk, the immersion time, the quantity of immersed fruit (approximately 300 g of fruit in 500 mL of immersion solution) and the variety of fruit (see Fruit section). Drying process (by convection)

After incorporating L. plantarum 299v in the fresh fruit, the product was subjected to a drying process. A pilot-scale tray dryer (Armfield, Ringwood, UK) was used because this equipment allows the dehydration of solid products by a flow of hot air. The drying process was performed with air at 40 °C and a speed of air flow of 1.5 m s1 (Rêgo et al., 2013). Drying of fruit with the adhered probiotic strain occurred in approximately 24–48 h, depending on the fruit. Afterwards, the dried fruits were ground with a laboratory mill to obtain a powder of probiotic fruit. Samples were taken at several stages of the process (after immersion, drying and grinding) for enumeration of viable cells of L. plantarum 299v.

© 2015 Institute of Food Science and Technology


(a)

(b)

Drying fresh fruit by convection

Immersion of fresh fruit in probiotic culture (Ringer’s or Skim Milk)

Grinding Fruit powders

+

Drying of fruit incorporated with probiotic by convection

Drying of probiotic culture by spray-drying (in skim milk)

Grinding of dried fruit with probiotic Figure 1 Schematic representation of the two methodologies used to obtain fruit powders with probiotic: (a) drying of the fruit with probiotic culture incorporated (by convection) and (b) drying of fruit (by convection) and addition of spray-dried probiotic culture.

Drying of fruit (by convection) and addition of spraydried probiotic culture Drying of fruit

Fresh fruits (apple, strawberry or banana) were dried by convection in a pilot-scale tray dryer, in the same conditions as described above, 40 °C and an air flow of 1.5 m s1. Dried fruit were ground with a laboratory mill to obtain powdered fruit. Drying of probiotic culture

The probiotic strain was dried by spray-drying. Lactobacillus plantarum 299v was grown in MRS medium, and cells were harvested by centrifugation and washed twice in sterile Ringer’s solution. Cells were resuspended to the original volume in skim milk (11% w/ v) obtaining a quantity of cells of 1010 cfu mL1 and then spray-dried in a laboratory scale apparatus (Niro Atomizer, Gladsaxevej, Denmark). Spray drier conditions were as follows: outlet air temperature 75 °C, inlet air temperature 200 °C and atomizing air pressure 4 Bar. Powder was collected in a single cyclone separator. At the end of the drying process, a probiotic powder was obtained with approximately 109 cfu g1. Addition of the probiotic culture to the dried fruit

After the processes of dehydration of both the fruit and the probiotic culture separately, the dried


Addition of dried probiotic to fruit powders (15% and 30%)

Fruit powders with probiotic

probiotic culture was added to the powdered fruit at a quantity of 15% and 30% (w/w of dry weight). These two percentages were used because with these quantities of dried probiotic culture were obtained food products with a suitable amount of cells (108–109 cfu g1) to be considered probiotic. Water activity of the fruit powders

The water activity of the powders was determined using a water activity meter (Aqualab, Series 3; Decagon Devices Inc., Pullman, WA, USA) at a constant temperature of 23 1 °C. Two readings were performed, and calibration was carried out with distilled water before the experiments. The fruits were dried until a water activity (aw) between 0.4 and 0.5 was achieved. Storage

Fruit powders with L. plantarum 299v, obtained in the two procedures described above, were stored in plastic containers at room temperature or at 4 °C for 3 months, in desiccators containing silica gel (aw = 0.03). The use of silica gel with a high water-absorbing capacity had previously been considered effective in maintaining the viability of dried cells during storage (Sudoma, 1990). Silica gel absorbents are


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available commercially and can be used in food packaging, because apart from being a powerful dehydrating agent and extremely efficient, silica gel is nontoxic and it is not expensive. For enumeration of viable cells of L. plantarum 299v, 1 g of each fruit powder was rehydrated in 9 mL Ringer’s solution and serial 10-fold dilutions were performed in Ringer’s solution. Samples were plated in duplicate on MRS agar using the Miles & Misra (1938) technique, and colony-forming units per gram (cfu g1) were determined after 48-h incubation at 37 °C. Results were expressed as log (N/N0), where N represents the cfu g1 of probiotic culture at several times during storage, and N0 represents the cfu g1 of probiotic culture in product immediately after processing. Statistical analysis

All experiments were carried out in duplicate. The results are expressed as mean standard deviation. An analysis of variance (ANOVA) was performed to assess any significant effects on the survival of L. plantarum 299v by the different methodologies used to produce the fruit powders with probiotic, as well as the temperature during storage. All calculations were carried out using the software Kaleidagraph (version 4.04, Synergy Software, Reading, PA, USA). Results and discussion

Drying process (by convection)

To develop apple, strawberry or banana powders enriched with probiotic by drying cubes of these fruits, an initial treatment was necessary to promote the incorporation of the probiotic strain. Lactobacillus plantarum 299v was incorporated in the fruits by immersion in a cell suspension, and then, fruits were dehydrated and ground. It is important to highlight that convective drying would be significantly less expensive than current methods used to produce fruit powders (e.g. freeze-drying). During this process, the viability of L. plantarum 299v decreased (Table 1).

After the incorporation step, the number of probiotic cells in the fruit samples was similar to values obtained in other studies incorporating probiotics by immersion (Ribeiro et al., 2014) or by vacuum impregnation (Betoret et al., 2003; Krasaekoopt & Suthanwong, 2008). In the drying process of strawberry and banana, only a slight or no reduction in probiotic cell viability was recorded (maximum of 1.0 log cfu g1). These data are consistent with a previous study which recorded a decrease in the probiotic viability of 1.0 log in these fruits during dehydration by hot air-drying (Ribeiro et al., 2014). In apple, reduction in probiotic viability was more pronounced, both during drying and during grinding processes (Table 1). It had been previously demonstrated that the production of dehydrated apple with probiotic by air-drying resulted in a reduction in the viable cell count of around two or three logarithmic cycles (Betoret et al., 2003; Rêgo et al., 2013). This reduction can be explained by the chemical composition of apple, such as the presence of organic acids and low pH values. It is known that malic acid is the dominant acid in apple and the variety Granny Smith has a high amount of malic acid (7.07 mg mL1) comparatively to other apple varieties, and in addition possess an acidic pH of approximately 3.40 (Wu et al., 2007). In this study, when the probiotic strain was previously resuspended in Ringer0 s solution, there was a reduction in the probiotic viability of more than 7.5 log cfu g1 after drying and grinding, while when cells were resuspended in skim milk, the reduction was ca. 2.6 log cfu g1 during the full processing. Therefore, in this assay, skim milk seems to have a protective effect and this medium could be a good solution to protect cells from dehydration and mechanical stresses. This protectant was selected because it can prevent cellular injury by stabilizing cell membrane constituents (Meng et al., 2008). In the other fruits, strawberry and banana, there was only a slight loss of cell viability, obtaining fruit powders with a suitable amount of probiotic, between 6 and 7 log cfu g1. Bacterial populations of 106–107 cfu g1 are established as an efficacious con-

Table 1 Viability of Lactobacillus plantarum 299v after immersion, drying (by convection) and grinding of fruit with incorporated probiotic culture (log cfu g1) Apple

Strawberry

Banana

Process

Ringer

Skim milk

Ringer

Skim milk

Ringer

Skim milk

Immersion* Drying Grinding

8.5 0.1a 3.4 0.1b 0.05). Furthermore, during the 3 months of storage, the probiotic in the strawberry powder showed a similar behaviour whether initially suspended in Ringer’s solution or skim milk (P > 0.05), with an observed reduction of 0.7 log cfu g1 and 0.9 cfu g1, respectively. In the banana powder, survival of the cells was significantly higher compared to the other fruits (P < 0.001). Lactobacillus plantarum 299v remained viable when stored at 4 °C as well as at room temperature. Thus, this matrix protected the probiotic cells throughout processing, unlike the other fruits processed. It was reported that fruit fibres can improve probiotic cells’ survival (Espırito Santo et al., 2012), and banana contains a higher quantity of dietary fibre (3.1 g per 100 g) compared to strawberry and apple (2.0 and 1.9 g per 100 g, respectively; Instituto Nacional de Sas´ de Doutor Ricardo Jorge, 2015). Also, during the storage of the banana powder, no significant differences were noted between the bacteria initially suspended in Ringer’s solution and the bacteria initially suspended in skim milk (P > 0.05). Water activity of fruit powders has an important outcome on probiotic survival during storage (Nualkaekul et al., 2012); in our study, the storage atmosphere was controlled by silica gel (water activity 0.03), so the effects of the initial suspending solutions may not be evident. The use of silica gel with a high water-absorbing capacity had previously been considered effective in maintaining the viability of dried cells during storage (Sudoma, 1990). Drying of fruit (by convection) and addition of spraydried probiotic culture

Figure 2 Viability of Lactobacillus plantarum 299v in apple (■), strawberry ( ) and banana ( ) powders dried by convection, after immersion of cell suspensions in Ringer’s solution (a) or skim milk (b). The viability was determined during storage at room temperature (solid line) and 4 °C (dashed line). All points are means standard deviations. Same letters indicate no significant differences (P ˃ 0.05).


An attempt was made to minimise losses in viability of the probiotic during the drying process, by combining the spray-dried probiotic culture with the fruit powders. Dehydration of probiotic or starter cultures by spray-drying is widely used to produce viable and active cultures, because this technology represents an economic approach with high production rates (Teixeira et al., 1995; Golowczyc et al., 2011; SalarBehzadi et al., 2013; Maciel et al., 2014). Viable counts of L. plantarum 299v after combining with the fruit powders were between 108 and 109 cfu g1 (Table 2). The viability of L. plantarum 299v when combined with the different fruit powders was evaluated during 3 months of storage (Fig. 3). In general, the viability of L. plantarum 299v decreased during storage, but it was not affected either by the quantity of probiotic added (15% or 30%) or by the storage temperature (4 °C or room temperature). The results indicate that cells could survive during storage at 4 °C or at room temperature in the presence of a powerful desiccant (dry silica


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Table 2 Cell viability of Lactobacillus plantarum 299v in fruit powders, when used the methodology of drying fruit by convection and addition of spray-dried probiotic culture at 15% and 30%. There is represented the probiotic viability (log cfu g1) immediately after processing and at the end of storage Apple

Strawberry

15% Initial time After 90 days (4 °C) After 90 days (room temperature)

30%

8.9 0.0 8.1 0.1b 7.7 0.2b a

15%

9.3 0.0 8.7 0.1b 8.5 0.1b

30%

8.0 0.0 7.5 0.0b 7.3 0.1b

a

Banana

a

15%

8.5 0.0 8.3 0.1b 8.0 0.0b a

30%

8.1 0.0 6.5 0.3b 6.4 0.0b a

8.2 0.0a 7.3 0.2b 7.0 0.0b

Same letters in the same column indicate no significant differences (P ˃ 0.05).

Figure 3 Viability of Lactobacillus plantarum 299v in apple (■), strawberry ( ) and banana ( ) powders dried by convection with added spray-dried probiotic culture at 15% (w/w) (a) and 30% (w/ w) (b). Viability was determined during storage at room temperature (solid line) and 4 °C (dashed line). All points are means standard deviations. Same letters indicate no significant differences (P ˃ 0.05).

gel), controlling water activity at 0.03. Storage conditions are critical factors for the stability of dried cells. It has been recommended to store dried cultures under vacuum, under controlled water activity or under dark conditions (Carvalho et al., 2004). Nevertheless, higher survival of the probiotic was recorded in the strawberry powder compared to the other fruit powders (P < 0.001), as shown in Fig. 3. These results are in agreement with Nualkaekul et al. (2012) where a higher survival rate of freeze-dried L. plantarum cells was reported when mixed with strawberry powder. In the banana powder, although in this second methodology we obtained a high amount of cells in the final product, it was observed a higher reduction


during storage comparatively to the first methodology. The drying process to which the probiotic cells were subjected to the two methodologies used in this study are different (drying by convection or by spraydrying); therefore, these techniques can have different consequences on cellular structure and, consequently, on its feasibility during storage. Thus, the thermal stress imposed by spray-drying may induce sublethal injuries on survivors and so, cells were less resistant to the storage conditions. Nevertheless, it would be necessary evaluate the cellular injuries after each process to confirm this concept. Overall, the cellular viability of L. plantarum 299v remains high during spray-drying and it is maintained during storage, resulting after 3 months of storage, in fruit powders with probiotic viable cell counts of between 106 and 108 cfu g1, as shown in Table 2. In this study, a higher survival rate was achieved using separate drying of fruit and probiotic cells, compared with the first process (drying of fruit infused with probiotic cells), so this procedure was shown to be more effective for the development of fruit powders enriched with L. plantarum 299v. However, these preliminary results should be complemented with further assays such as the nutrient composition of the fruits after process, the ability of L. plantarum 299v to survival in a simulated gastro-intestinal tract and sensory analysis. Conclusion

To develop a functional food product that combines the beneficial characteristics of fruits and probiotic cultures, fruit powders enriched with L. plantarum 299v were produced. The final product could be used in the food industry, for example, to produce natural juices or as an ingredient in numerous desserts. Several factors can affect the survival of a probiotic culture, such as the food processing stages and storage conditions. The methodology that combines separate drying techniques (hot air and spray-drying, for the fruit and probiotic, respectively) proved to be the best way to obtain the proposed food product. Thereby, the final



product contained a level of probiotic cells, deemed efficacious in probiotic products. The fruit powders with probiotic can be stored at 4 °C or room temperature under a controlled low relative humidity environment. Acknowledgment

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