Concentrated whey from block freeze concentration or milk-based ice creams on Bifidobacterium BB-12 survival under in vitro simulated gastrointestinal conditions

Abstract

This study’s main highlight refers to the utilization of concentrated whey, obtained from freezing concentration processes in ice cream manufacturing. The second important point refers to the comparison between dairy and whey concentrated ice creams’ Bifidobacterium BB-12’s protective effects under in vitro simulated gastrointestinal conditions. Both ice cream types showed bifidobacteria viable cell counts higher than the recommendation for a product to be considered a probiotic. The incorporation of such bacteria into both ice cream varieties showed the most important probiotic characteristic, which is survivability. Bifidobacterium BB-12 demonstrated survival rates higher than 90% after the upper digestive tract passage into the colon. In addition to that, the best protective effect was pointed out from whey-based ice cream testing. Considering such statements, the addition of probiotic bacteria and concentrated whey on ice cream manufacturing could be decisive when producing functional foods, providing benefic effects on the consumer.

Keywords:  block freeze concentration; probiotic ice cream; whey; concentration; gastrointestinal simulation

1 Introduction

Ice cream has been described as a complex frozen colloid, consisting of a continuous aqueous phase with ice crystals dispersed in a semi-frozen solution (Akbari et al., 2019; Saremnezhad et al., 2020). The original ice cream formulation consists on milk and, in some cases, functional elements. Replacing milk with concentrated whey obtained through freezing concentration processes was not yet described on ice cream production.

Cheese whey is a liquid obtained from precipitated casein removal in cheese-making processes (Remón et al., 2016). According to Hausjell et al. (2019), around 190 million tons of cheese whey is generated yearly worldwide, causing great environmental impact. The volume of effluents produced by the cheese industry is directly proportional to its market demand. Considering mass production, one kilogram of cheese requires roughly 10 kilos of milk and yields nine kilograms of whey. Cheese whey is a byproduct that represents significant environmental impact due to its physicochemical composition, comprehending β-lactoglobulin, α-lactalbumin, immunoglobulin, serum albumin and lactoferrin. On the other hand, such effluent is rich in nutrients and cannot be simply discarded into the environment without adequate treatment (Prazeres et al., 2012; Hausjell et al., 2019). Due to its low solids rate, concentration techniques can be applied to facilitate its usage and further processing. As an example, thermal evaporation under vacuum systems is typically employed and results in undesirable sensorial and nutritional changes in the byproduct (Menchik & Moraru, 2019). Thus, liquids’ non thermal concentration processes, including block freeze concentration, have been gaining interest in the past few years. This process is based on water’s fractional crystallization into ice and its sequential separation from the concentrated liquid through gravitational thawing (Petzold et al., 2015). Petzold et al. (2015) reported advantages on the process, such as a simple separation step and sensible food components reduction. Block freezing concentration is considered an environmentally friendly technology, with high effectiveness and low energy consumption. Also, it preserves the product’s sensorial and nutritional qualities and doesn’t require expensive maintenances (Aider & Halleux, 2008). Muñoz et al. (2018a) stated that the energy required on freezing concentration processes is relatively low when compared to evaporation and other water removal techniques.

The usage of concentrated whey instead of milk on fermented milk beverages’ production was successfully reported by Canella et al. (2018). Silva et al. (2015) stated that, despite the fact that dairy production represents one of the most developing segments and a major branch of the functional food industry, further studies are required on functional ice creams. Probiotics are considered functional, since they present live microorganisms that provide health benefits, such as intestinal microbiota maintenance, when consumed regularly (Hill et al., 2014). Bifidobacterias are the most common probiotic species within normal human intestinal tract (Hadad et al., 2019). The probiotic strain Bifidobacterium animalis subsp. lactis BB-12 (BB-12) shows benefic effects on human’s gastrointestinal health and immune system (Liu et al., 2020). Some studies have pointed ice cream as a great probiotic bacteria carrier (Champagne et al., 2015; Akalin et al., 2018; Balthazar et al., 2018; Calligaris et al., 2018; Góral et al., 2018), however, Kozłowicz et al. (2019) defends that the ice cream composition can lead to serious damages on probiotic bacteria cells. Thus, probiotic microorganisms should survive not only during food processing, but also through gastrointestinal flow (Soares et al., 2019). The integrity of the cell membranes, intracellular pH and functional enzymes of probiotic cells can be constantly challenged by stressful factors such as gastric acidity, biliary acids and digestive enzymes during their way through the digestive tract (Rodrigues et al., 2019). Therefore, studies should focus on how probiotic cells respond to all in vitro gastrointestinal conditions. Gastric and biliary acids play an important role on body defenses against ingested microorganisms, killing and controlling gastrointestinal exposure to many pathogens. However, this same defense mechanism can also disable potentially beneficial microbes, such as probiotics. Considering probiotic effects that depend on the viability and intestinal physiological activity, the probiotic’s survival in gastric and biliary acids of the upper gastrointestinal tract is a critical point (Jungersen et al., 2014). Therefore, the usage of concentrated whey instead of milk in ice cream elaboration was reported as a suitable way to develop functional ice creams. This study’s objective was to enhance concentrated whey’s usage on probiotic ice creams production, considering its high availability and lower cost, as well as evaluating bifidobacterial survivability to different in vitro simulated gastrointestinal environments.

2 Material and methods

2.1 Materials

Two ice creams were manufactured using 61.70 g/100g of pasteurized whole milk (12.03 g/100g of total solids) (Papenborg, Biguaçu, Brazil) and concentrated whey (18.61 g/100g of total solids) obtained from block freeze concentration process at its second stage, respectively. The block freeze concentration process is based on the complete freezing of whey, followed by a partial defrosting procedure under simple gravitational separation. Once the whey was frozen, 50% of the initial volume was defrosted at room temperature (20 ± 2 °C), resulting in two fractions denoted as concentrated whey and ice. The composition of both ice creams 1 (made of whole milk) and 2 (made of concentrated whey) consisted on 16.03 g/100g of cream with 45 g/100 g of fat content (Tirol, Treze Tílias, Brazil); 0.06 g/100g of sodium chloride (Ciasal, Areia Branca, Brazil) and; 22.21 g/100 g of sucrose (Camil, São Paulo, Brazil). A probiotic strain of Bifidobacterium BB-12 (Bifidobacterium animalis subsp. lactis, Chr. Hansen, Honsholm, Denmark) was added at end of the process. In vitro simulated gastrointestinal conditions required the usage of the following enzymes: α-amylase (28.75 U/mg protein), pepsin from porcine gastric mucosa (400 U/mg protein) and pancreatin from porcine pancreas (digestive power - 8 X USP specifications), as well as bovine bile salt (28.75 U/mg protein), all purchased from Sigma-Aldrich (St. Louis, MO, USA). An appropriate medium containing the major compounds present in the colon (large intestine) was prepared using bacteriological peptone, yeast extract, tryptone and Tween 80, acquired from Oxoid (Hampshire, UK). To mimic colon conditions, NaCl, KH₂PO₄, MgSO₄·7H₂O, KCl, citrus pectin, casein, starch, and L-cysteine from Sigma-Aldrich (St. Louis, MO, USA) were used. The usage of MRS broth (Difco, Sparks, USA) provided an excellent base for bacterial strain behavior comparison. It was used as a control sample after the exposition to in vitro simulated gastrointestinal conditions, as well as during the preparation of bifidobacteria suspensions. MRS agar (Difco, Sparks, USA), sodium propionate (Fluka, Neu-Ulm, Germany), lithium chloride (Vetec, Rio de Janeiro, Brazil) and AnaeroGen (Oxoid, Hampshire, UK) were used to determine bifidobacterial viable cell counts.

2.2 Preparation of bifidobacteria’s suspensions

Bifidobacterium BB-12’s freeze-dried culture was rehydrated in sterile skimmed milk, accordingly to Fritzen-Freire et al. (2012), named stock solution, and stored in sterile bottles at - 18 ± 2 °C. Then, bifidobacteria’s cell suspension was prepared accordingly to Rodrigues et al. (2011), along with modifications. The stock solution was added to the MRS broth, modified with 0.2 g/100 g of lithium chloride and 0.3 g/100 g of sodium propionate, and then submitted to incubation in anaerobic jars with AnaeroGen® at 37 ± 1 °C for 48 h. After incubation, bifidobacteria cells were harvested by centrifugation (1,000 x g) (centrifuge from Nova Técnica, São Paulo, Brazil) at temperature equal to 25 ± 1 °C during 10 min. The supernatant was discarded and the cells were washed twice with a saline solution (0.85/100g). Bifidobacteria cells were suspended in the following solutions: 20 mL of MRS broth; 20 mL of whole pasteurized milk and; 20 mL of concentrated whey. Final Bifidobacterium BB-12 viable cell counts in each suspension were 9.81 log CFU/mL (colony-forming units per mL), 10.04 log CFU/mL, and 9.64 logs CFU/mL, for MRS broth (control), whole milk and concentrated whey, respectively.

2.3 Ice cream production

The ice cream initial mix consisted on pasteurized whole milk (ice cream 1) and concentrated whey, obtained from freeze concentration process’ second stage (ice cream 2). Cream, sodium chloride and sucrose were then added to both ice creams, in quantities previously described in section 2.1. Both mixes were pasteurized at 75 ± 2 °C during 30 min at water bath (Model DI950M, Dist, Florianópolis, SC, Brazil); and cooled down to 4 ± 1 °C. Posteriorly, bifidobacteria’s cell suspensions (20 mL/L) were added into the ice creams. Ice creams were produced in batch freezers (Model ICE 21, Cuisinart, New Jersey, USA), stored in 100g plastic containers and then hardened in the freezer before further analysis, providing three samples.

2.4 In vitro simulated gastrointestinal experiment

In vitro simulated gastrointestinal experiments were performed in triplicates, simulating typical conditions of different body parts such as the human mouth, esophagus, stomach, duodenum, ileum and colon sequentially, as established by Barretta et al. (2019) and as demonstrated in Figure 1. In the colon scenario, samples were collected at 12 h (ascending colon), 24 h (transverse colon) and 48 h (descending colon). The in vitro experiments were performed with 25 g of both 1 and 2 ice cream samples. As well as the ice creams samples, 25 mL of the MRS broth (control) along with 20 mL/L of bifidobacteria’s suspension was also submitted to in vitro experiment. Thus, ice cream 1, ice cream 2 and control (MRS broth) samples were evaluated under in vitro gastrointestinal conditions. Prior to its usage, all enzyme solutions were filter-sterilized through 0.22 µm membrane filters (MF-Millipore, Billerica, MA, USA) and maintained on ice baths during the simulation. The temperature (37 ± 1 °C) and peristaltic movement intensity of the human body were simulated water baths (Model DI950M, Dist, Florianópolis, SC, Brazil). As it occurs in natural gastrointestinal conditions, the overall volume increased under in vitro gastrointestinal conditions.

Figure 1
Protocol used during gastrointestinal in vitro digestion phases.

2.5 Bifidobacterium BB-12 viable cells count

At the end of each simulated gastrointestinal stage, samples were evaluated on bifidobacteria viable cell counts. Each sample dilution was corrected with peptone water (0.1 g/100 g) to 225 mL/25 g of each ice cream or 225 mL/25 mL of MRS broth. Samples were blended in a bag mixer 400 (Interscience, St. Nom, France), serially diluted with peptone water (0.1 g/100 g) and plated in triplicates on MRS agar with lithium chloride (0.2 g/100 g) and sodium propionate (0.3 g/100 g), as described by Vinderola & Reinheimer (2000). Plates were incubated in anaerobic jars with AnaeroGen® at 37 ± 1 °C for 72 h and Bifidobacteria viable cell counts were carried out in triplicate and expressed as log colony-forming units per gram (log CFU/g).

2.6 Survival rate

Bifidobacteria’s performance under in vitro conditions was calculated in survival rate (Equation 1), as described by Guo et al. (2009).

$\text{Survival rate (\%)=}\frac{{\text{N}}_{\text{1}}}{{\text{N}}_{\text{0}}}\text{×100}$ (1)

where N₁ represents Bifidobacterium BB-12 viable cell counts (log CFU/g) after exposure to each simulated in vitro gastrointestinal stage and N₀ is bifidobacteria viable cell counts (log CFU/g) before such stages.

2.7 Data analysis

All data was expressed considering mean ± standard deviation of the triplicate measurements of three samples for each lot. Statistical analysis was applied through analysis of variance (ANOVA), followed by Tukey’s test. Statistical significance was attributed when P < 0.05. Results were processed in STATISTICA 13.3 (TIBCO Software Inc., Palo Alto, CA) software.

3 Results and discussion

Figure 2 and Table 1 shows Bifidobacterium BB-12 viable cell counts and its survival rate after being submitted to each in vitro simulated gastrointestinal stage, respectively. Both ice cream samples could be considered a good vehicle for Bifidobacterium BB-12, since the probiotic viable cell count was higher than the recommended amount for a product to be considered a probiotic. Hill et al. (2014) affirmed that a given probiotic might induce several health-promoting factors if the probiotic viable cell count is ≥ 6 log UFC/g or mL, enhancing gastrointestinal homeostasis by positively affecting the intestinal microbiota balance. Akalin et al. (2018) and Kozłowicz et al. (2019) classified the ice cream as a great product to be used as a probiotic vehicle because of its stability in frozen systems and its composition that includes proteins, fat and lactose. Kozłowicz et al. (2019) also stated that the ice cream pH is higher than fermented milk’s, which has a positive impact on probiotic bacteria’s survival. Soares et al. (2019) confirmed that species of the genus Bifidobacterium are notoriously more sensitive to acid (4.5-5.0) and alkaline pH (8.0-8.5) foods. Silva et al. (2015) affirmed that the aeration and freezing of the ice cream mix can lead to serious damage to bacterial cells. However, these authors also stated that the use of an appropriate inoculum amount of the probiotic bacterial strain can contribute to high bacterial abundance. Other factors involved in maintaining probiotic viable cell counts could be related to the ice cream mix composition. Rodrigues et al. (2019) reported that milk is currently the main probiotic vehicle used by the food industry worldwide. Silva et al. (2015) verified that goat’s milk ice cream was an adequate delivery vehicle for bifidobacteria.

Figure 2
Bifidobacterium BB-12 viable cells count data mean from (a) control sample (MRS broth); (b) ice cream 1 sample, prepared with whole milk; and (c) ice cream 2 samples, using concentrated whey, after exposure to each one gastrointestinal in vitro digestion phase. The bars represent the standard deviation (I).

Table 1
Bifidobacterium BB-12 survival rate (%) results expressed as mean ± standard deviation, after exposure to each gastrointestinal in vitro phase from control (MRS broth), ice cream 1 (with whole milk), and ice cream 2 (with concentrated whey) samples.

Phase		Sample
		Control	Ice cream 1	Ice cream 2
Mouth		98.36 ± 0.90cB	92.63 ± 2.46dC	101.85 ± 0.70bA
Esophagus/Stomach		96.34 ± 0.22dC	97.88 ± 0.68bB	100.03 ± 0.86b,cA
Duodenum		97.40 ± 0.99c,dB	95.17 ± 0.44c,dC	99.67 ± 0.86cA
Ileum		97.73 ± 0.52cB	96.96 ± 0.98b,cB	101.03 ± 0.84b,cA
	Ascending	111.08 ± 0.56aB	103.85 ± 3.31aC	112.79 ± 0.68aA
Colon	Transverse	109.11 ± 1.04a,bB	107.25 ± 3.04aB	112.04 ± 1.67aA
	Descending	109.25 ± 0.13bB	106.56 ± 0.39aC	112.57 ± 0.74aA

• ^A,B,C
  Within a row, different superscript uppercase letters denote significant differences (P < 0.05) between samples among the same step of the gastrointestinal in vitro phase. ^a,b,c,d Within a column, different superscript lowercase letters denote significant differences (P < 0.05), for the same sample among the different steps of the gastrointestinal in vitro phase.

Madureira et al. (2011) affirmed that probiotic bacteria’s behavior in MRS broths provides an excellent base for comparison, evaluating different ice cream compositions’ protective effects. Therefore, in this study, it was verified that ice cream 2 samples (made of concentrated whey) demonstrated to be a great probiotic vehicle. Yasmin et al. (2019) also observed a protective effect of Bifidobacterium longum BL-05 in simulated gastrointestinal conditions, using whey proteins mixes. These authors concluded that such protective effect was due to the abundance of specific amino acid residues from whey that provides a protective environment for probiotics. Etchepare et al. (2020) noted that the buffering effect presented by higher whey protein presence contributed to higher rates of probiotics survival during gastrointestinal tests. These authors also observed that the use of whey proteins provided substantially better protection for probiotic bacteria Lactobacillus acidophilus La-14. Therefore, it was expected that, if probiotics are incorporated into the food system, they should remain stable during the processing of the food until consumption. Furthermore, it was also expected that orally ingested probiotics should survive the harsh acidic condition of the gastrointestinal tract, overcoming the upper digestive tract to reach the colon. For this reason, this study aimed on evaluating Bifidobacterium BB-12’s survival rate under in vitro simulated gastrointestinal environments, when incorporated into milk and concentrated whey ice cream mixes. The addition of Bifidobacterium BB-12 into the ice creams and in vitro processing was realized sequentially, accordingly to the established by our research group. However, in the present work, the innovation is also related to further studies on colon’s background, where samples were collected after 12 h (ascending colon), 24 h (transverse colon) and 48 h (descending colon).

The passage through the gastrointestinal tract includes different challenges for bifidobacteria added into MRS broth, ice cream 1 and ice cream 2 samples. Overall, it was noticed bifidobacteria’s survival rates remained high (> 90%) (Table 1) on all samples and through mouth, esophagus and stomach, duodenum and ileum stages. High Bifidobacterium BB-12’s survival rates found in all samples could be related to this microorganism’s gene coding for bile salt hydrolase, an enzyme responsible for coping with high bile salt concentrations in the small intestine. This enzyme is always present and active in Bifidobacterium BB-12, as described by Jungersen et al. (2014). Sánchez et al. (2007) stated that studies with Bifidobacterium BB-12 indicated several genes involved in fatty acid biosynthesis were down-regulated in the presence of bile salts and suggested that bile salts could induce changes in bifidobacteria’s cytoplasmic membrane composition. These authors also revealed that bifidobacteria’s metabolic reorganization is also correlated to changes in the preferential branch through glycolytic pathway, and hence in the manner of energy production. Sánchez et al. (2007) also cited others changes of bifidobacteria which are associated with its resistance after exposure to gastrointestinal conditions, such as the occurrence of the fatty acid biosynthesis; the increase of molecular chaperones amount; the modification of cell ‘s redox balance and; the nitrogen metabolism, which could be involved with a sigma factor. Zhang et al. (2019) reported that the sigma factor is responsible for the major gene transcription that allows bacteria to respond to environmental changes. According to these authors, sigma factors are multi-domain subunits of bacterial RNA polymerase, which plays vital roles in the transcription initiation process and has great effects on many important cellular activities, such as growth and stress tolerance, considering environmental changes. However, it should be pointed out that the best bifidobacteria survival rate’s performance (P < 0.05) was found in ice cream 2. This confirms that, even though well-equipped to endure this critical passage in the gastrointestinal tract, Bifidobacterium BB-12 may suffer several damages, resulting in a survival rate decrease, as verified by Muñoz et al. (2018b). As in our study, these authors also identified a better bifidobacteria protective effect in soft fresh cheese from freeze concentrated milk, and therefore, containing more cheese whey protein when compared to MRS broth. This leads to believe that the increase of total solids due to the whey proteins in the ice cream sample was responsible for a more stable and protective food matrix for bifidobacteria. Tamime et al. (2006) reported that the addition of whey proteins increases the buffering capacity, contributing to a better survival rate of probiotic microorganisms. Because these authors emphasized that the freezing stress during ice cream production must be considered as a limiting factor for probiotic survival rate, we also consider that bifidobacteria’s protective effect is related to the concentrated whey used on the elaboration of ice cream 2.

Just like the probiotic’s survival in the presence of gastric acid and bile from the upper gastrointestinal tract is critical, it is also crucial for bifidobacteria’s survival in the colon (large intestine). Jungersen et al. (2014) affirmed that the human large intestine presents a wide variety of bacteria, with bifidobacterias being prominent members of this complex ecosystem. Presumably, Hadad et al. (2019) stated that the reduction in oxygen levels from colon stages enables anaerobic bacteria like bifidobacteria to grow. This was confirmed in this study and is demonstrated by the highest (P < 0.05) bifidobacteria survival rate (Table 1) detected in colon stages (ascending, transverse, descending). Shu et al. (2020) observed an increase in probiotic survival rate when the environment changes went from strong acid to a suitable one (pH ≥ 6). These same authors assert that the cause of this increase is associated with an acidic environment. In this case, probiotics strengthen its cell walls and the cell membrane permeability changes. Therefore, the H+ is isolated outside of the cells, protecting them, which is an important reaction promoted by bacteria in acidic environments considering DNA’s damage repair and acid-induced protein (Shu et al., 2020). It is noteworthy that probiotic effects depend on the intestinal viability and physiological activity after the probiotic passage and survival against gastric and biliary acids from upper gastrointestinal tract. Therefore, the use of concentrated whey obtained by a non-thermal and cheaper process (block freeze concentration) as a Bifidobacterium BB-12 matrix protection increased the ice cream’s functional value, since this specific probiotic bacteria showed a higher survivability under in vitro simulated gastrointestinal environments in comparison to milk-based ice cream.

4 Conclusion

Both milk and concentrated whey ice creams presented high amounts of bifidobacterial viable cell counts after exposure to in vitro simulated gastrointestinal environments, overtaking the recommendation for probiotic products. The Bifidobacterium BB-12 added to the ice creams showed the most important characteristic of probiotic microorganisms, demonstrating the ability to survive through the upper digestive tract to reach the large intestine. Concentrated whey ice creams showed the best bifidobacteria protective effect after exposure to in vitro simulated gastrointestinal backgrounds, demonstrating great tolerance to environmental changes. Also, the higher total solids content of concentrated whey is mostly represented by proteins.

Acknowledgements

The authors are grateful to CNPq (National Council for Scientific and Technological Development, Brazil) for the financial support [CNPq, 405965/2016-8], and to CAPES (Coordination of Improvement of Higher Education Personnel, Brazil - Finance Code 001) by the scholarship.

• Practical Application: Usage of concentrated whey’s instead of milk in to develop functional ice creams.

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Publication Dates

• Publication in this collection
  05 Jan 2022
• Date of issue
  2022

History

• Received
  15 Sept 2021
• Accepted
  22 Oct 2021