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Article

A Model for Probiotic Fermented Food Production

by
Maria Luisa Savo Sardaro
1,2,3,*,
Silvia Zaini
2 and
Katherine Ryan Amato
1
1
Department of Anthropology, Northwestern University, Evanston, IL 60208, USA
2
Department of Food and Drug, University of Parma, 43121 Parma, Italy
3
Department of Human Science and Promotion of the Quality of Life, University of San Raffaele, 00166 Rome, Italy
*
Author to whom correspondence should be addressed.
Appl. Sci. 2023, 13(20), 11123; https://doi.org/10.3390/app132011123
Submission received: 24 July 2023 / Revised: 7 September 2023 / Accepted: 3 October 2023 / Published: 10 October 2023
(This article belongs to the Special Issue Microbiology of Fermented Foods and Beverages: Analysis and Applications)
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Abstract

:
The past few decades have demonstrated how important the human gut microbiota is for human health. Because of this, the use of microbiota-modulating dietary interventions such as probiotics and prebiotics is growing in popularity amongst consumers, food manufacturers, healthcare professionals, and regulators. In particular, there is interest in making a wider variety of foods with probiotic properties. However, as a solution for food manufacturers to produce fermented foods compatible with the “probiotic foods” label definition, we used an impedometric analysis to identify the survival and growth capacity of microbial strains in specific environmental contexts. Using this approach, manufacturers can more effectively select the strains with the highest growth rate for use in probiotic fermented food production trials. To provide a proof of concept, we tested three Lacticaseibacillus rhamnosus probiotic strains growing in milk at different temperatures. We quantified the probiotic’s growth using species-specific primers and quantitative real-time PCR. Overall, our results demonstrate the feasibility of this type of model in facilitating the production of probiotic fermented foods by allowing manufacturers to select strains able to grow under specific conditions. Our model can be used to develop, increase, and target the beneficial health properties of a multitude of fermented foods produced worldwide.

1. Introduction

Research on the gut microbiota (GM) over the past two decades has shown that it functions like another organ with important influences on human health and likely evolution [1,2,3,4,5]. A healthy GM has been shown to be responsible for improving the digestion of substances such as fiber and is also significantly involved in the production of functional metabolites and substances necessary for life, such as amino acids, vitamins, and short-chain fatty acids (SCFAs) [6,7,8,9,10]. Numerous studies have reported complex connections between diet, the GM, and human health [3,11,12,13,14,15], including diet-mediated effects on metabolic function, pathogen resistance, immune system development, and regulation of gastrointestinal functions and associated diseases (e.g., food allergies, gastrointestinal inflammation, neurodegenerative disease, and tumors) [10,16,17,18,19]. Many of these effects are likely a result of the interaction between bacteria and host intestinal epithelial cells (IECs), which is considered central for the regulation of immunity at the host–microbe interface [19,20], affecting the host immune response [19,20].
Given that environmental factors such as diet can strongly influence the composition and function of the GM, there has been widespread interest in developing microbial interventions for a range of diseases and health risks. Probiotics, or “live microorganisms which when administered in adequate amounts confer a health benefit on the host” [21,22,23], are becoming more common. A variety of strains have been shown to have beneficial effects, such as the mitigation of food allergies, the prevention of diarrhea, reducing cholesterol, and immunomodulation [24,25,26,27]. However, probiotics alone are often not enough to permanently shift the GM [28]. Prebiotics or fibers that feed microbes are necessary to support probiotic strains. As a result, food-based interventions with both probiotic and prebiotic traits are likely to be the most effective.
Fermented foods are historically important parts of the modern human diet, and it has been hypothesized that they were a critical part of our ancestors’ diets [1]. In addition to their nutritional benefits, fermented foods have both live bacteria and prebiotic properties [29,30,31,32] because they are commonly produced using the fermentation pathways of lactic acid bacteria (LAB), such as Lactobacillus and Bifidobacterium. Their consumption has been associated with numerous health benefits [30,33,34,35,36,37,38]. For example, Lactobacillus can reduce the duration or frequency of respiratory and gastrointestinal infections [39,40,41] via secretion of anti-pathogen substances, competitive exclusion of pathogens, maintenance of mucosal integrity, and stimulation of systemic or mucosal immune responses [42]. However, these health impacts can vary depending on how a given fermented food is produced, and while epidemiological evidence demonstrates broad health benefits, such as the ability to reduce disease risks and enhance longevity and quality of life [43,44,45], few random controlled trials (RCTs) or prospective studies have been undertaken to pinpoint specific health benefits from specific foods and bacterial strains, combined with the relatively low amount of beneficial bacteria in most fermented foods, most manufacturers cannot label their foods as ‘probiotic products’ [21]. The terms “fermented food” and “probiotics” cannot be used interchangeably, and “probiotic” should only be used when there is a demonstrated health benefit conferred by well-defined and characterized live microorganisms [21]. This distinction is challenging for most fermented food producers, given the cost of RCTs and the large number of experiments required to confirm the health benefits of the probiotic bacteria contained in their fermented foods, even without strain-specific evidence. As a result, most food manufacturers are limited to using the term “Contains probiotics” on their labels.
To address this gap and simultaneously facilitate the production of validated ‘fermented foods’ by manufacturers and the consumption and diffusion of fermented foods in the public, we establish a model approach for assessing and augmenting the microbial content of common fermented foods. The goal is to provide food manufacturers with a cost-effective and powerful tool to integrate the already positive characteristics of most fermented foods [30,36,46,47,48,49] with the specific health-promoting attributes of certified probiotic strains. Not only will this allow manufacturers to better design experiments and clinical trials to leverage ‘probiotic’ labels, but it will likely lead to more health benefits for consumers.
We use dairy culture as a proof of concept since fermented milk and dairy products are considered dietary staples around the world, and beyond their nutritional and organoleptic properties, their health benefits have long been known [48,50,51,52]. Previous in vitro cheese studies using a probiotic L. rhamnosus HN001 showed beneficial effects, including the increased availability of calcium, magnesium, and zinc, together with dipeptides, tripeptides, free amino acids, and free fatty acids. These molecules stimulate gastrointestinal responses by increasing the activity of digestive enzymes, colonocyte proliferation and, consequently, ion transport [53,54,55]. We tested our model by evaluating the survival and growth capacity of three Lacticaseibacillus rhamnosus probiotic strains in milk and dairy products. We wanted to determine the extent to which we could use this approach to increase the number of microbial CFUs (colony-forming units) to the certified dose associated with a “probiotic food” label.
The ability to identify multiple probiotic strains that can be used in a given fermented food can facilitate the production of multi-strain probiotic fermented products that also fulfill the health goal of maintaining and increasing gut microbial diversity [4,56,57,58]. For example, the cardiovascular health benefit associated with Bacillus subtilis in natto [49,59,60] can be combined with the cholesterol-lowering effect of Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus plantarum or Lactobacillus reuteri [61,62] by screening for their ability to survive and grow together in the specific conditions dictated by natto ingredients and fermentation conditions. Using the approach demonstrated here, our overall goal is to stimulate the application of this model in the large-scale production of probiotic fermented products characterized by different thermal treatments, sources of raw material, and aging conditions typical of many commercial fermented foods already largely known and widely consumed (Kefir, cheeses, labne, feta, sauerkraut, pickles, olives, kimchi, gochujang, ssamjang, doenjang, natto, miso, etc.).

2. Material and Method

2.1. Bacteria Strains

We tested the capacity of three commercial Lacticaseibacillus rhamnosus probiotic strains: L. rhamnosus LR06 (DSM 21981) [63], L. rhamnosus LR04 (DSM 16605) [64], and L. rhamnosus HN001 [65] to survive and grow in a milk and cheese matrix. Strains were obtained from Probiotical SpA (Novara, Italy) and DuPont Danisco (Italy). All the strains were maintained under lyophilized conditions and as stock cultures at −80 °C in De Man, Rogosa and Sharpe (MRS) broth (Oxoid, Basingstoke, UK) supplemented with 20% (v/v) glycerol until use. The stock cultures were revitalized before usage by inoculating 200 µL of thawed cultures in 6 mL of sterile MRS incubated for 24 h at 37 °C in anaerobiosis condition. Overnight cultures were counted to verify the microbial cell load, washed with Ringer solution (Oxoid, Basingstoke, UK) and properly diluted to reach an inoculum level of 6 Log CFU/mL.

2.2. Confirmation of Lyophilized Probiotic Bacteria Concentration by Plate Count Method

One gram of the three lyophilized probiotic L. rhamnosus strains was homogenized thoroughly in 99 mL of sterile Ringer solution (Merck 1.15525) and further tenfold serially diluted for the determination of the viable bacteria using standard pour plate procedures. The MRS agar plates were incubated at 37 °C for 3 days under anaerobic conditions to standardize the amount of lyophilized probiotics to use in cheese production.

2.3. Acidification Ability of the Commercial Probiotics L. rhamnosus Strains in Milk

The ability of the three Lacticaseibacillus rhamnosus probiotic strains to grow in and acidify milk was investigated with impedometric analysis using a Bactrac 4300® (Sylab, Generon, San Prospero, MO, Italy) [66]. The BacTrac 4300® system measures two specific impedance values: the E-value, which refers to the impedance change at the electrode surface, and the M-Value, which is the change in conductivity in the medium. This variation in the electrical conductivity of milk is proportional to the change in the number of microorganisms and their metabolic activity. Therefore, the microbial growth in milk can be measured using the corresponding M-Value [67]. The commercial probiotic strains tested were diluted after being revitalized to a final level of 6 Log CFU/mL in sterile Ringer solution and used to inoculate 36 mL of pasteurized milk. The total amount was equally divided into three sterilized BacTrac 4300® vials and incubated. The impedance measurement was performed at two incubation temperatures: 37 °C for 72 h or 52 °C for 30 min, followed by 37 °C for 72 h. The ability to acidify was quantified using M-values, which represent the overall impedance variation of the media in the vials. The M-value was recorded every 10 min for 72 h and shown as M%. This value is automatically calculated by the instrument as a relative electrical change compared to a starting value. The pH of the milk was measured electrometrically at the end of the 72 h of incubations with a Beckman ϕ™ 300 series pH meter (Beckman Instruments, Inc. 4300 N. Harbor Blvd., Fullerton, CA, USA). All the analyses were carried out in triplicates. Negative samples, consisting of non-inoculated milk, were also incubated for each temperature tested.

2.4. DNA Extraction

DNA extraction was performed from 1 mL of milk or 1 g of cheese dissolved in Trisodium Acetate 2%, and placed at 50 °C for 30 min. After centrifugation at 9500 rpm for 10 min and the removal of the fat portion, the pellet was resuspended in Buffer 1 and Lysozyme (Sigma-Aldrich, Milan, Italy), followed by a 30 min at 37 °C. Subsequently, the QIAamp DNA Stool Mini Kit (Qiagen, Milan, Italy) manufacturer’s protocol has been followed. The presence and quality of the extracted DNA were evaluated using 1% (w/v) agarose gel electrophoresis.

2.5. Real-Time PCR

We used a commercial probiotic strain, Lactobacillus rhamnosus GG (ATCC 53103), as a reference strain for the real-time PCR standard curve preparation. In this study, 12.5 mg of lyophilized L. rhamnosus GG was diluted in 1 L of pasteurized milk (an inoculum level of 6 Log CFU/mL), and five subsequent serial dilutions were performed in 20 mL of a solution of pasteurized milk (10-8-6-4-2 mL) and trisodium citrate at 2% (Sigma Aldrich) (10-12-14-16-18 mL, respectively). The lyophilised product suspension was centrifuged (3500× g, 10 min), and DNA was extracted from the pellet by using the QIAamp DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol. The five dilutions from L. rhamnosus GG genomic DNA preparations were amplified using real-time PCR.
After the PCR reaction optimization, the standard curve was established using the serial dilutions of L. rhamnosus GG samples from the initial concentration of 106 CFU/mL of viable bacterial cells to simulate the cheese product inoculation. Aliquots of the standard samples prepared for DNA extractions were plate counted. Based on standard curves, the reaction efficiencies were determined, and the possible inhibition of PCR reactions was examined.
The correlation between Ct values and CFU/mL was determined by using Design and Analysis Software v2.6, QuantStudio (Thermo Fisher, Monza, Italy).
The standard curves’ parameters obtained by the fivefold dilution samples series showed an R2 = 0.9925, an amplification efficiency of 109.64%, and a slope of −3.1206.
The absolute quantification of the L. rhamnosus strains (HN001 and LR06) was performed on milk samples before inoculation as negative controls, on samples collected right after inoculation with 106 CFU of probiotic, on the discarded whey samples, in cheese after 3 days of inoculation and in cheese after one month of ripening.
PCR amplifications were performed in a 25 µL reaction volume containing SYBR™ Green PCR Master Mix (Applied Biosystems, Monza, Italia), 0.2 µM of each primer, and 1 µL of genomic DNA extract. The primers used were L. rhamnosus specific, PoxProm FW ‘TGAAAGGGYTTGCATTGTTAT’ and PoxProm RV ‘AATGCGCCYACTTCTTCATG’ [68]. The PCR amplification was performed using a QuantStudio™ 3 Real-Time PCR (ThermoFisher). Bacterial detection was achieved with a 3-step qPCR with the initial denaturing step of 50 °C for 2 min and 95 °C for 2 min, 40 cycles of 95 °C for 30 s, 60 °C for 15 s, 72 °C for 20 s, and then 95 °C for 1 min and 55 °C for 30 s. All samples were also subjected to melting curve analysis in the temperature from 60 to 95 °C with an increase of 0.5 °C for 1 s to establish the specificity of the amplification. DNA from the L. rhamnosus GG bacteria was included in the PCR standard curve assay. For the DNA quantification, triplicate samples were used, and the mean of CFU (colony-forming unit) quantity per gram wet weight was calculated, also considering the correspondence with the plate count for the possible presence of multiple putative pox genes [69].
C o p i e s / g = D N A   c o n c e n t r a t i o n   ( n g / µ L ) × 6.02 × 10 23   m o l e c u l e s / m o l e s 660 g / m o l e × t a r g e t   D N A   l e n g t h   ( b p ) × 1 × 10 9   n g / g

2.6. Probiotic Fermented Food Model

An initial milk culture trial was performed to identify the probiotic strain able to survive the thermal treatment process (temp range from 37 °C and 52 °C) typical for dairy products and grow most efficiently in the first month of ripening. The probiotic strains tested were L. rhamnosus LR06 and L. rhamnosus H001, selected via impedometric analysis. Lactobacillus helveticus commercial strains (Alce srl, Novara, Italia) were also used with a dilution of 2 g/L, as is typical in cheese production.
The survival and growth of the two probiotic L. rhamnosus strains, inoculated in milk at inoculum amounts of 106, were monitored by using absolute real-time PCR quantification in the acidified curd and in the cheese after 1 month of ripening at 18 °C, and the experiments were performed in triplicate.

2.7. Statistical Analysis

Ct values were automatically generated by using QuantStudio 3 software v1.5.1. One-way analysis of variance (ANOVA) with post hoc Tukey test and pairwise comparisons was used to compare the results obtained through the use of impedometric analysis and quantitative real-time PCR. Linear regression analysis to determine the statistical significance between the probiotic cheese products was performed by using RStudio v2022.07.1 version (RStudio, PBC, Boston, MA, USA).

3. Results and Discussion

3.1. Growth and Acidification Ability of L. rhamnosus Strains in Milk

The growth and acidification capacity of the three probiotic strains were tested under two different temperatures, 37 °C and 52 °C, to demonstrate the efficacy of the impedometric test in screening multiple bacteria strains both for their ability to grow in a specific ingredient and at different technological temperatures. For example, dairy milk products like yogurt and kefir have an optimal temperature of up to 37 °C [70]. The same probiotic strain can also survive at higher temperatures of 52 °C for a limited amount of time, leading to the production of some types of raw milk cheeses. Traditional aged raw milk hard cheeses such as Swiss Emmentaler PDO, Parmigiano Reggiano PDO, Cheddar, Grana Padano PDO, and Emmentaler PDO typically have a scalding condition, a sub-pasteurization heat treatment applied for the manufacture, which can vary between 52 and 55 °C × 30–40 min [71,72,73].
The impedometric analysis shown in Figure 1 revealed significant variation among the strains in their ability to grow in milk (Tukey multiple comparisons of means, p < 0.005). The probiotic strains HN001 and LR06 showed a higher acidifying capacity associated with an increase in conductivity (M-value), meaning that they were able to grow, metabolize and acidify milk. At the same time, they showed different adaptation behaviors to the temperatures and milk environments. The HN001 strain reached maximum exponential growth, with an M-value of 30 at 30 h, both at the sub-optimal temperature and at the stress temperature, with no significant differences between the two conditions (p = 0.9). On the other hand, the LR06 strain showed an extended Lag phase, reaching the highest M-value of 30 at 50 h under the stress conditions of 52 °C and after 70 h at the sub-optimal temperature of 37 °C (p = 0.39). Despite the LR006 strain showing a longer Lag phase, it was able to reach the same level of acidification as strain HN001 under the stress condition, with no significant differences among the two strains at the end of the incubation (p = 0.9). In contrast, the strain LR04 showed a very limited capacity to grow in milk at both temperatures, reaching an M-value max of 10 at the end of incubation.
The impedometric results were also confirmed via pH evaluation, in which the HN001 and LR06 strains showed the highest acidity level with a pH value of 3.7 ± 0.2 and 3.5 ± 0.1, respectively (Table 1); strain LR04 showed a limited reduction in pH values compared to the control sample under stress conditions with a pH value of 6.7 ± 0.2 versus 7.2 ± 0.1. At 37 °C, the pH of LR04 was higher than the control, reaching 7.3 ± 0.5. The ability to lower the pH is an important food safety quality in fermented products since it is linked to the production of organic acids with important antimicrobial features that inhibit spoilage and pathogen bacteria growth [24,74]. Based on the impedometric findings together with the pH values, the two strains, LR06 and HN001, were chosen for further trial studies to test their capacity to grow and acidify in the cheese environment.

3.2. Evaluation of the Capacity to Grow during Cheese-Making Process

The HN001 and LR06 strains were evaluated for their ability to survive and grow during the cheese-making process for up to one month of ripening using absolute quantification via real-time PCR before inoculation, immediately after inoculation, in discarded whey, and at three days and one month after inoculation. The investigated samples were treated with the same extraction method, and the Ct value for each sample was calculated using the standard curve to obtain the bacterial content (shown as log10 copies of bacterial DNA per gram; Figure 2). All the analyses were performed in triplicate. As we can observe in Figure 2, strain HN001 showed a slightly higher adaptation to cheese technological processes, showing a slightly faster growth rate and a higher concentration after one month of ripening compared to strain LR06 (2.8 × 108 CFU/mL versus 1.5 × 108 CFU/mL, respectively). However, the difference was not significant (p = 0.46).
Both L. rhamnonus strains (HN001 and LR06) showed the ability to survive and grow in the thermal and nutritional conditions typical of dairy cultures, providing the possibility to use them as adjunct probiotics in a multitude of fermented products characterized by a similar environment. Also, the use of more probiotic strains simultaneously could offer the possibility, as in this case, to combine the immune support and allergy prevention benefits associated with the HN001 probiotic strain with the ability of the LR06 strain to restore the gastric barrier [64,75,76,77]. Moreover, the ability to carry out a large probiotic screening based on thermal treatment tolerance and the ability of following probiotic growth using real-time PCR can help promote the production of probiotic fermented foods characterized by beneficial effects, such as aged dairy products with long fermentation–ripening times in which the glycolysis is enhanced, and lactose is exhausted by LAB and other lactic acid microorganisms. This process also creates the necessary sensory characteristics of fermented foods thanks to longer lipolysis and proteolysis activities that influence not only the generation of myriad of flavor compounds, their precursors, and the SCFAs [78] but also the rheology and external appearance by softening its texture.
With our model, we strive to promote the production and diffusion of a large variety of probiotic fermented products from vegetables, beverages, dairy and non-dairy fermentation. Our model uses impedometric evaluation as a first step to obtain a list of probiotic strains able to grow in the specific conditions dictated by the product ingredients. The next step is using quantitative real-time PCR to identify the initial inoculum amount of probiotics for the subsequent fermentation process. The inoculum amount is affected by the different sources of nutrients, the length of the fermentation and aging, and the relative serving size of the product. Considering that the adequate amount of probiotics per serving is generally reported to be higher than 109 CFU [79]; it is important to identify a specific initial inoculum amount that fulfills the probiotic food labeling requirements. An example is reported in Figure 3, where the growth of L. rhamnosus HN001 probiotic strain has been tested in whole milk under temperature stress conditions, with two different inoculum amounts, 103 CFU/mL and 106CFU/mL. Under both conditions, we could confirm, through the use of real-time PCR, the rapid growth of the probiotic strain that reached, at one month, the values of 3.8 × 107 CFU/mL and 1.03 × 108 CFU/mL, respectively (p value < 0.005). These results demonstrate that only the initial inoculum amount of 106 CFU/mL fulfills the requirements of probiotic dairy products characterized by a short ripening time, confirming the importance of testing the inoculum amount.
Using the findings from our model, we hypothesize that vegetable fermented products (e.g., natto, sauerkraut, pickles) with fermentation times less than one month and with relatively low sugar content need to have a bigger inoculum (~107 CFU/g) to reach an adequate amount per serving size after a month (e.g., 45 g for natto or 30 g sauerkraut) (Figure 4); this hypothesis is supported by the LAB growth curve reported by Ghimire et al. in the natural fermentation of Gundruk, a Himalayan fermented vegetable dish [80]. In contrast, fermented beverages (bread and fruit kvass, pulque, tejuino, water kefir, kombucha) [81,82,83,84,85,86,87] characterized by larger serving sizes (~100 g) and greater sugar content can have a smaller inoculum (~106 CFU/g). Milk cultures with a large serving size (e.g., yogurt, kefir, sour cream, labne) or with a smaller serving size (~25 g) but a ripening period of less than six months due to high sugar content and high bacterial growth should be similar [88,89]. The production of probiotic fermented food products with longer fermentation times (>6 months) could be more challenging, especially for those with fermentation times longer than 12 months, due to the decline in bacterial growth over time as a result of water and nutrient limitation [90]. In these cases, it might be more ideal to use a lower amount of the initial inoculum to be able to maintain adequate growth over the longer ripening time.

4. Conclusions

In this study, we set out to develop a cost-effective and powerful model to augment the probiotic characteristics of fermented foods. The model is designed to promote the production of probiotic fermented foods by using probiotic certified strains to make fermented food. It first uses impedometric evaluation to test multiple probiotic strains simultaneously and efficiently for their ability to grow in any fermented raw ingredient and in different technological conditions, such as temperature. Subsequently, real-time qPCR helps to identify the most suitable probiotic strains for each fermented product by quantifying bacterial growth in the different aging and environmental conditions characteristic of different types of fermented foods.
The model has some limitations in the context of natural starters that are characterized by large, undefined cultures. However, these can be overcome with the development of multiplexing qPCR that can discriminate closely related probiotic strains. Currently, though, our model is a feasible replacement for other commonly used techniques in industry. For instance, other genotypic identification techniques rely on the time-consuming isolation of the target strain using selective culture media. These approaches limit the types of specific probiotic strains used in fermented food production. In contrast, our model can be considered an effective tool, facilitating the development of new fermented food products with probiotic features for food producers and providing consumers with a wider variety of probiotic food products to incorporate into their diet.

Author Contributions

Conceptualization, M.L.S.S. and K.R.A.; methodology, M.L.S.S. and S.Z.; software, M.L.S.S.; validation, M.L.S.S. and S.Z.; formal analysis, M.L.S.S. and S.Z.; investigation, M.L.S.S. and S.Z.; writing—original draft preparation, M.L.S.S.; writing—review and editing, M.L.S.S. and K.R.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partially funded by the Emilia-Romagna Region, Italy (PARENT project POR-FESR 2014–2020). Project Parent, Siteia-Parma (2016–2017), concluded and disseminated in April 2018 (http://parent.crpa.it/nqcontent.cfm?a_id=17191&tt=t_bt_app1_www, URL accessed on 5 October 2023).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available on request by contacting the authors.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 13 11123 g001
Figure 1. Impedometric evaluation of the three L. rhamnosus strains (HN001, LR04, LR06) in milk at 37 °C and 52 °C.
Figure 1. Impedometric evaluation of the three L. rhamnosus strains (HN001, LR04, LR06) in milk at 37 °C and 52 °C.
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Figure 2. Absolute quantification of L. rhamnosus HN001 and LR06 strains via qPCR in the one-month dairy culture. “Milk” milk, “Milk_PB” milk added of probiotic, “Whey” whey samples, “0_month” Milk after 3 days of inoculation, “1_month” milk after one month of inoculation.
Figure 2. Absolute quantification of L. rhamnosus HN001 and LR06 strains via qPCR in the one-month dairy culture. “Milk” milk, “Milk_PB” milk added of probiotic, “Whey” whey samples, “0_month” Milk after 3 days of inoculation, “1_month” milk after one month of inoculation.
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Figure 3. Absolute quantification of L. rhamnosus HN001 strain via qPCR in one-month dairy cultures. “Milk_PB” milk added of probiotic, “0_month” Milk after 3 days of inoculation, “1_month” cheese after one month of inoculation, by adding 103 CFU/mL (blue) and 106 CFU/mL (red) of L. rhamnosus HN001 strain.
Figure 3. Absolute quantification of L. rhamnosus HN001 strain via qPCR in one-month dairy cultures. “Milk_PB” milk added of probiotic, “0_month” Milk after 3 days of inoculation, “1_month” cheese after one month of inoculation, by adding 103 CFU/mL (blue) and 106 CFU/mL (red) of L. rhamnosus HN001 strain.
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Figure 4. Probiotic fermented food model.
Figure 4. Probiotic fermented food model.
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Table 1. pH values of the milk cultured with the three probiotic L. rhamnosus strains.
Table 1. pH values of the milk cultured with the three probiotic L. rhamnosus strains.
Skim Milk 37 °C × 72 hpH
HN0013.7 ± 0.1
LR063.5 ± 0.2
LR047.3 ± 0.5
Negative control7.1 ± 0.2
Skim Milk 52 °C × 30 min 37 °C × 72 h
HN0013.7 ± 0.2
LR063.5 ± 0.2
LR046.7 ± 0.2
Negative control7.2 ± 0.1
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Savo Sardaro, M.L.; Zaini, S.; Amato, K.R. A Model for Probiotic Fermented Food Production. Appl. Sci. 2023, 13, 11123. https://doi.org/10.3390/app132011123

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Savo Sardaro ML, Zaini S, Amato KR. A Model for Probiotic Fermented Food Production. Applied Sciences. 2023; 13(20):11123. https://doi.org/10.3390/app132011123

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Savo Sardaro, Maria Luisa, Silvia Zaini, and Katherine Ryan Amato. 2023. "A Model for Probiotic Fermented Food Production" Applied Sciences 13, no. 20: 11123. https://doi.org/10.3390/app132011123

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