Using β-galactosidase and Novel Probiotic Strains to Formulate Lactose-Free Acidophilus Milk Fortified with Cocoa Powder

doi:10.21203/rs.3.rs-1758600/v1

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Research Article

Using β-galactosidase and Novel Probiotic Strains to Formulate Lactose-Free Acidophilus Milk Fortified with Cocoa Powder

https://doi.org/10.21203/rs.3.rs-1758600/v1

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The study was investigated to develop the lactose-free probiotic acidophilus milk (PAM) enriched with novel probiotic strains (Lactobacillus acidophilus LDMB01, Lactococcus lactis LDMB10, and Streptococcus thermophilus LDMB09), as well as fortified with cocoa powder. The physicochemical, microbiological, and sensory analyses were performed to see how the formulations worked. Two probiotic acidophilus milk formulations were made with β-galactosidase lactose hydrolysis, one with cocoa (5% m/v). A constant sugar level of 5% was used. The lactose-free acidophilus milk did not differ in acid content and showed values below 0.1% for lactose. The incorporation of cocoa in the PAM provided higher initial pH values reduced with the storage period and higher syneresis values throughout 28 days of storage. In addition, the probiotic bacteria showed high viability during storage, with counts above 7 log CFU/mL. In terms of sensory evaluation, most judges assigned both products an overall acceptability score of 8 for the characteristics (appearance, aroma, taste, texture, and overall acceptance). On the other hand, the sample containing cocoa achieved higher mean scores for appearance and texture. As a result, these products have much potential for industrial use, especially for lactose-intolerant people.

Food Science & Technology

Cocoa powder

lactose-free

Lactobacillus acidophilus

fermented milk

sensory acceptance

Dairy products, particularly fermented products, have risen in popularity in recent decades due to their health-promoting properties, leading to their classification as nutraceuticals, functional foods, or designer foods [1]. As a result, new versions are being developed to fulfill consumer needs. These items contain a single or many bacterial species contributing to the products' favorable properties [2, 3]. Probiotic bacteria are used to make various fermented milk [4]. Different probiotic bacteria like the genera of Lactobacillus and Bifidobacterium have been used to make other fermented dairy products. These microbes have various health benefits, including maintaining the gastrointestinal tract's balance [5]. Prebiotics has been successfully introduced into several lactose-containing dairy products such as Greek yogurt [6], whey beverage [7], yogurt [8], mousse [9], and fermented milk to boost the action of probiotics and increase fiber consumption [10]. Probiotics have got much interest for their nutritional and functional features, and various research has been conducted to figure out how they work in the human body [11]. As a result, several health organizations recommend a daily dose of viable probiotic cells per serving portion (10⁹ CFU/day). Probiotic cultures have been added to frozen desserts to expand the variety of probiotic foods available on the market [12].

All over the world, about 66–70% of adults have lactose malabsorption, which means they cannot break down lactose [13]. When lactose-containing foods are consumed, intestinal digestion and colonic fermentation are affected, which can cause diarrhea and other health problems. On the market are lactose-free products made from hydrolyzed or alternative milk sources that lack nutritional and sensory value [14]. Compared to lactose-containing products, these products will cost you more money. As a result, there is much demand for lactose-free products that are nutritious, cost-effective, and well-liked by people [14]. Dairy desserts are popular among many consumers, including kids and the elderly [15], primarily due to their appealing nutritional and sensory characteristics [16, 17].

The changes to acidophilus milk composition serve various purposes, including improving nutritional value, enhancing probiotic survival during storage, stimulating acidification by probiotics, and improving sensory properties without incurring high costs to meet consumers' growing needs and health expectations [18–20]. Today, the focus is more on how food can keep people healthy and well-being and avoid certain diseases than on how it can satisfy their hunger or give them nutrition. Products with health claims, like probiotic foods, are being made because people are more concerned about their health because of scientific knowledge about how diet and health work together. To promote excellent health and illness prevention, a wide array of tastes, additives, and probiotic cultures that enhance the flavor and texture of the food are incorporated [21]. However, numerous obstacles exist to producing high-quality probiotic products, including the accumulation of acids, oxygen, and peroxide levels and unanticipated culture interactions [19]. As a result, it is preferable to fortify it with various chemicals if necessary to combat such an unpleasant flavor [22].

Based on the background mentioned above, this research aimed to provide a very initial approach to formulate probiotic acidophilus milk using pre-hydrolyzed milk using β-galactosidase, investigating the fortification effect of cocoa powder to develop probiotic acidophilus milk, and evaluating the lactose-free probiotic acidophilus milk through physicochemical, microbiological and sensory analysis.

The current research work was carried out at the Dairy Microbiology and Biotechnology Lab, Department of Dairy Science, and the Interdisciplinary Institute for Food Security Lab, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

2.1 Preparation of the probiotic acidophilus milk

Pasteurized bovine milk (3% fat, Milk Vita, Bangladesh) was hydrolyzed with β-galactosidase (0.15% v/v) (Merk, Germany) at 38°C for 2 h [23]. The chemical composition of milk used in the product manufacturing is shown in Table 1. Two types of fermented milk were prepared according to Fig. 1: (i) probiotic acidophilus milk (PAM - without flavor) and (ii) PAM added cocoa with flavor 5% (m/v). Both fermented milks were added with 5% (m/v) sucrose. The milk was separately heated at 90°C for 15 min and promptly cooled to 42°C. Afterward, the milk culture composed of Lactobacillus acidophilus LDMB01, Lactococcus lactis LDMB10 and Streptococcus thermophilus LDMB09 [24] (from Dairy Microbiology and Biotechnology Lab, Bangladesh Agricultural University) were inoculated (1% m/v) to give a concentration of about 6 log CFU/mL of probiotic bacteria in milk. The milk samples were incubated at 42°C until pH 4.6 reached (about 4 to 5 h). After fermentation, the products were cooled, gently stirred, packed in glass containers, sealed with aluminum foil lids, and stored at 4 ± 1°C. The microbiological and physicochemical studies were carried out in triplicate on storage days 1 and 28, respectively, while the sensory analysis was carried out after 7 days of storage.

Components	Raw bovine milk
Components	Before enzyme treatment	After enzyme treatment (Before fermentation)
Total solids (%)	12.32 ± 1.12	12.65 ± 0.94
Fat (%)	3.00 ± 0.02	3.10 ± 0.03
Protein (%)	3.52 ± 0.11	3.54 ± 0.04
Lactose (%)	4.10 ± 0.62	0.92 ± 0.02
Acidity (%LA)	0.11 ± 0.00	0.12 ± 0.01
pH	6.89 ± 0.03	6.75 ± 0.02

2.2 Physicochemical characterization

The levels of lactose were determined in hydrolyzed and fermented milk using a milk analyzer (Lactoscan, Bulgaria), according to Pinto et al. [25]. The pH of the samples was measured using a pH meter (Hanna, Bulgaria). Acidity (% lactic acid) was determined by titrating with 0.1 mol/L NaOH. The viscosity of the fermented probiotic milk (PAM) with or without cocoa samples was measured at 25°C using a digital Viscometer (Brookfield, USA), according to the method of Islam et al. [26]. The spindle number LV 2 was used, and the spindle was rotated at 0.5 rpm. At the end of the 15th second of the measurement period, the readings were noted as centipoises (cP). The susceptibility to syneresis (STS) was measured following the method of Isanga and Zhang [27]. In short, 100 g of prepared acidophilus milk sample was placed in a funnel lined with a Whatman filter paper (no. 1). After drainage for 6 h at room temperature, the volume of whey kept in a beaker was measured and calculated STS by using the following formula:

STS (%) = V₁/V₂ × 100

Where, V₁ = volume of whey collected after drainage; V₂ = volume of yogurt sample.

2.3 Microbiological analyses

The strains Lactococcus lactis LDMB10 was enumerated using MRS agar (Himedia, India), modified by adding 0.2% (m/v) lithium chloride (Himedia, India) and 0.3% (m/v) sodium propionate (Sigma-Aldrich, USA), while L. acidophilus LDMB01 was enumerated using MRS agar added with an overlay of the same medium, as Vinderola and Reinheimer [28] described. The enumeration of S. thermophilus was performed using M7 agar (Himedia, India) supplemented with lactose solution (10% m/v) [29].

2.4 Sensory analysis

The tasting panelists comprised students and faculties of Bangladesh Agricultural University, provided with the evaluation guidelines before judging. Panelists of 20 people (10 male, 10 female) aged between 20–45 years comprised the panel to evaluate the sensory profile of acidophilus milk. The appearance, smell, flavor, texture, and overall acceptance were evaluated through a structured hedonic scale of 9 points (1-dislike extremely; 9-like extremely) [30]. Each judge received 50 mL of each sample at 5 ± 1°C, presented in a monadic sequential way, served in plastic cups encoded with random three-digit numbers. Also, water was used to clean the judges’ palates between each evaluation.

2.5 Statistical analysis

The data were evaluated by analysis of variance (ANOVA) followed by Tukey's test (P < 0.05) using SPSS 17.0 for Windows statistical package (SPSS Inc., Chicago, 149 IL, USA).

3.1 Physicochemical characteristics

The data regarding pH, acidity, syneresis, lactose content, and viscosity for the probiotic acidophilus milk (PAM) are shown in Table 2.

3.2 Acidity and pH

There was no difference in acidity between the samples but varied on days 1 and 28 of storage (p < 0.05). According to Vénica et al. [31], because of changes in the buffering capacity of the milk bases, the greater concentrations of galactose, glucose, and sucrose in sweetened hydrolyzed PAM may have an inhibitory effect on the generation of lactic acid due to suppression of lactose metabolism and undesirable osmotic effect. As a result, a lower product post-acidification was regarded a technological benefit. The pH values of probiotic acidophilus milk with or without cocoa were not significantly different (P > 0.05). This parameter revealed that after 28 days of storage, the cocoa sample had a lower pH (P > 0.05) than the initial stage. Meanwhile, both samples revealed a reduction in pH during storage, impacting the syneresis index. The absence of traditional yogurt starter bacteria, especially L. bulgaricus, and the use of L. acidophilus as the sole fermenting organism along with other adjuncts probiotic culture kept the pH and acidity of both types of samples consistent during storage. In the presence of starter cultures, fermentation can continue during refrigeration, resulting in post-acidification and a subsequent decrease in yogurt pH [32–34].

Table 2

Titratable acidity, pH, lactose, syneresis and viscosity index of lactose-free probiotic acidophilus milk on day 1 and day 28 at 4°C.
Characteristics	Storage	PAM without cocoa	PAM with cocoa
Titratable acidity (% LA)	1	0.81 ± 0.03^aA	0.80 ± 0.02^aA
Titratable acidity (% LA)	28	0.92 ± 0.05^aB	0.91 ± 0.07^aB
pH	1	4.41 ± 0.12^aA	4.42 ± 0.08^aA
pH	28	4.33 ± 0.04^aB	4.32 ± 0.07^aB
Lactose (%)	1	0.09 ± 0.05	0.12 ± 0.01
Lactose (%)	28	0.04 ± 0.06	0.06 ± 0.02
Syneresis (%)	1	5.13 ± 0.02^bB	7.52 ± 0.37^aB
Syneresis (%)	28	14.16 ± 0.32^bA	20.52 ± 0.04^aA
Viscosity (cP)	1	14834.62 ± 51.23^bB	17456.28 ± 53.27^aB
Viscosity (cP)	28	18022.62 ± 44.23^bA	21421.20 ± 41.27^aA

Values are expressed as mean ± standard deviation (n = 3). Within a row, different lowercase letters indicate significant differences between samples (p < 0.05). Within a column, different uppercase letters indicate significant differences between storage periods (p < 0.05). PAM = probiotic acidophilus milk.

3.3 Lactose

The hydrolyzed milk (before fermentation) had a lactose percent of < 1.0% (Table 1), but after fermentation, the samples showed lactose values < 0.1% (Table 2). These results agreed with the values established by the Brazilian legislation [35] and European Food Safety Authority [36]; therefore, these products could be considered lactose-free.

3.4 Syneresis

In this study, the addition of cocoa influenced syneresis of PAM (p < 0.05). Syneresis was significantly lower in plain PAM than in cocoa-supplemented fermented milk (Table 2). Syneresis of plain yogurt was 5.13%, whereas after adding cocoa, it became 7.52% at the fresh state of the product. However, the product's syneresis was increased in the storage period and reached 14.16% in PAM without cocoa and 20.52% with cocoa-supplemented samples (Table 2). The observed behavior is consistent with Pereira et al. [37], who studied the effect of adding cocoa on lactose-free symbiotic milk. Furthermore, Meyer et al. [38] reported that plant fiber molecules could bind water, preventing their free movement. Also, cocoa interacts with milk proteins, providing stability to the protein network, and thus originates a protein matrix less prone to whey expulsion.

3.5 Viscosity

The viscosity of the PAM with or without cocoa differed significantly (p < 0.05). In this study, the initial viscosity of plain PAM was 14834.62 cP; after adding cocoa, the value was increased to 17456.28 cP. On day 28 of storage, the viscosity rate was shifted to 18022.62 cP and 21421.20 cP for PAM without cocoa and supplemented cocoa, respectively. The reason behind it might be that the water loses during the storage period. However, according to Marshall [39], Martin-Diana et al. [40], and Isanga and Zhang [27], a higher viscosity can be obtained if the fermented milk contains a higher level of fat.

3.6 Microbiological analyses

The results regarding probiotic cell viability of the lactose-free probiotic acidophilus milk samples are presented in Fig. 2. All three microbes were higher than 6 log CFU/mL on days 1 and 28 of storage, characterizing the fermented milk as probiotics. Throughout storage, both lactose-free fermented milk formulations had viable cell counts of Lactococcus lactis LDMB10 above 7 log CFU/mL, indicating that the lactose-free probiotic acidophilus milk provided a suitable environment for the survival of the probiotics, and there was no difference (p > 0.05) in counts between storage days in the plain sample but significantly differed (p < 0.05) in cocoa fortified sample during the storage period. The L. lactis LDMB10 count in the plain PAM was marginally lower than in the cocoa sample on day 28 of storage. In the case of the L. acidophilus LDMB01 cells, there were few variations across samples (p > 0.05) at the latter storage stage. This microbe had high viability (> 8 log CFU/mL). When comparing the PAM sample to the cocoa sample, a greater count (p < 0.05) of S. thermophilus was seen on the first day of storage in the cocoa fortified sample. The viability of these bacteria was slightly higher in the cocoa sample throughout the storage period. The L. lactis LDMB10 count in the plain PAM was somewhat lower on day 28 of storage than in the cocoa sample. L. lactis LDMB10, according to Canella et al. [10], may aid probiotic survival during dairy product preservation. Therefore, further studies are necessary to establish the behavior of this strain in lactose-free fermented milk during storage and in vitro digestion. There were minor changes across samples or storage days in the case of the L. acidophilus LDMB01 cells. This microbe had a high vitality (> 7.5 log CFU/mL). Vénica et al. [41] found similar results after examining the viability of the LA-5 strain in fermented acidophilus milk containing cocoa derived from lactose hydrolysis for 28 days. According to those authors, the matrix's carbohydrate composition or cocoa's presence in the fermented acidophilus milk did not affect microbial viability. After 180 days of storage at 4°C, Laliˇci ́c-Petronijevi ́c et al. [42] observed that probiotic L. acidophilus NCFM® outlived Bifidobacterium lactis HN019. These probiotic bacteria were L. acidophilus, L. casei, L. paracasei, and B. lactis. Begum et al. [43] evaluated the viability of Lactobacillus acidophilus NIAI L-54 in whey-based watermelon juices stored at 4°C for 21 days. In addition, the same strain was used in a probiotic beverage using whey and pineapple juice, and the viable probiotic cell counts were approximately 5.0 log CFU/mL up to 56 days of refrigeration storage [44]. They discovered that the viability was only 4 log CFU/mL, which they attributed to the food matrix utilized [45]. In continuation, Islam et al. [46] found that the isolated new strains of Lactiplantibacillus plantarum from raw goat milk showed the high survivability in 1% bile salt with potential cholesterol-lowering activity.

3.7 Sensory evaluation

The acceptability test results for the plain PAM and PAM with cocoa samples are shown in Fig. 3. Most assessors evaluated the samples based on a 9-point hedonic scale for both samples. From Fig. 2, it was shown that there was a significant difference (p < 0.05) in several sensorial properties between the two samples of PAM and PAM with cocoa. The aroma, body and texture, and taste of cocoa were smoother and more delicate than plain PAM. There were no variations between the samples regarding overall acceptance, smell, or flavor. These findings demonstrate that judges were more accepting of cocoa flavor samples. The current investigation revealed that both samples had high sensory acceptance levels, providing a new option for the industry to address consumer demand for functional lactose-free products. Despite the significant incidence of lactose intolerance among the population, the variety of lactose-free products is still limited, particularly in the case of products containing probiotics and prebiotics. Numerous scholars have examined the sensory profile of fermented products such as acidophilus milk. Generally, a sample will be considered acceptable if the mean value of 70% of panelists is more than 5. The panelists evaluated the product's taste, color, appearance, flavor, and general acceptance [47]. Additionally, it was tested on fermented milk containing varying amounts of green banana pulp, with 6 g green banana pulp added to 100 g fermented milk being the most acceptable amount. Concentration resulted in a decrease in humidity, an increase in solid fat content, and a reduction in texture score. The post-storage acidification effect decreased acceptability, implying the need for stabilizers or buffers during production to offset the post-storage acidity effect [20]. Similarly, a study indicated that coconut milk added with banana pulp and skimmed milk powder was typically acceptable, resolving the issue of excessive flavor and texture [22]. The grape pomace extract to goat milk improved the product's color, flavor, and overall acceptability [48].

The probiotic acidophilus milk made with cocoa flavor displayed satisfactory physicochemical, microbial, and sensory qualities in the current investigation. Throughout the product's shelf-life, the viable count of L. acidophilus remained above 6 log CFU/mL. So, the samples had high probiotic strain viability and high sensory acceptability scores in color, appearance, aroma, taste, body texture, and overall acceptability. On the other hand, the sample was lactose-free because the lactose level was less than 0.1%. However, the sample with cocoa received higher sensory values for appearance, taste, aroma, and feel. More research is needed to determine how these lactose-free fermented milk functions in the human body. These findings might assist the dairy business in diversifying market possibilities, particularly for lactose-intolerant people.

CRediT authorship contribution statement

Md. Zakirul Islam: Conceptualization, Writing original draft, Investigation, Formal analysis. Sharmin Akhter: Sample processing and analysis. Ruckshana Islam Liza: Review & Grammatical proof. Md. Shahriar Islam Sojib: Analysis, Draft writing & Editing. Md. Sayed Hasan: Draft writing. Raihan Habib: Co-supervision. Md. Harun-ur-Rashid: Supervision and Formal investigation.

Conflict of Interest

The authors declare that there is no conflict of interest.

Acknowledgment

The authors are thankful to the Ministry of Science and Technology, the People's Republic of Bangladesh, for the financial support in this project.

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Using β-galactosidase and Novel Probiotic Strains to Formulate Lactose-Free Acidophilus Milk Fortified with Cocoa Powder

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Abstract

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1. Introduction

2. Materials And Methods

2.1 Preparation of the probiotic acidophilus milk

2.2 Physicochemical characterization

2.3 Microbiological analyses

2.4 Sensory analysis

2.5 Statistical analysis

3. Results And Discussion

3.1 Physicochemical characteristics

3.2 Acidity and pH

3.3 Lactose

3.4 Syneresis

3.5 Viscosity

3.6 Microbiological analyses

3.7 Sensory evaluation

4. Conclusion

Declarations

References

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