Mechanistic study of the differences in lactic acid bacteria resistance to freeze- or spray-drying and storage.

Lactobacillus delbrueckii subsp. bulgaricus and Lactiplantibacillus plantarum are two lactic acid bacteria (LAB) widely used in the food industry. The objective of this work was to assess the resistance of these bacteria to freeze- and spray-drying and study the mechanisms involved in their loss of activity. The culturability and acidifying activity were measured to determine the specific acidifying activity, while membrane integrity was studied by flow cytometry. The glass transitions temperature and the water activity of the dried bacterial suspensions were also determined. Fourier transform infrared (FTIR) micro-spectroscopy was used to study the biochemical composition of cells in an aqueous environment. All experiments were performed after freezing, drying and storage at 4, 23 and 37 °C. The results showed that Lb. bulgaricus CFL1 was sensitive to osmotic, mechanical, and thermal stresses, while Lpb. plantarum WCFS1 tolerated better the first two types of stress but was more sensitive to thermal stress. Moreover, FTIR results suggested that the sensitivity of Lb. bulgaricus CFL1 to freeze-drying could be attributed to membrane and cell wall degradation, whereas changes in nucleic acids and proteins would be responsible of heat inactivation of both strains associated with spray-drying. According to the activation energy values (47-85 kJ/mol), the functionality loss during storage is a chemically limited reaction. Still, the physical properties of the glassy matrix played a fundamental role in the rates of loss of activity and showed that a glass transition temperature 40 °C above the storage temperature is needed to reach good preservation during storage. KEY POINTS: • Specific FTIR bands are proposed as markers of osmotic, mechanic and thermal stress • Lb. bulgaricus CFL1 was sensitive to all three stresses, Lpb. plantarum WCFS1 to thermal stress only • Activation energy revealed chemically limited reactions ruled the activity loss in storage.

An Apple and Acáchul Berry Snack Rich in Bioaccessible Antioxidants and Folic Acid: A Healthy Alternative for Prenatal Diets.

A fruit leather (apple and acáchul berry) oriented toward women of reproductive age was developed. The snack was supplemented with an ingredient composed of folic acid (FA) and whey proteins (WPI) to ensure the required vitamin intake to prevent fetal neural tube defects. In order to generate a low-calorie snack, alternative sweeteners were used (stevia and maltitol). The fruit leather composition was determined. Also, an in vitro digestion process was carried out to evaluate the bioaccessibility of compounds with antioxidant capacity (AC), total polyphenols (TPCs), total monomeric anthocyanins (ACY), and FA. The quantification of FA was conducted by a microbiological method and by HPLC. The leather contained carbohydrates (70%) and antioxidant compounds, mainly from fruits. Bioaccessibility was high for AC (50%) and TPCs (90%), and low for ACY (17%). Regarding FA, bioaccessibility was higher for WPI-FA (50%) than for FA alone (37%), suggesting that WPI effectively protected the vitamin from processing and digestion. Furthermore, the product was shown to be non-cytotoxic in a Caco-2 cell model. The developed snack is an interesting option due to its low energy intake, no added sugar, and high content of bioactive compounds. Also, the supplementation with WPI-FA improved the conservation and bioaccessibility of FA.

Sensory, physicochemical, and functional stability of freeze-dried blackcurrant (Ribes nigrum L.) Snacks. Effect of sweeteners.

The objective of this work was to develop two freeze-dried snacks from blackcurrant and different sweeteners: honey/isomalt (HI) sweetened with honey/isomalt, and isomalt/stevia (IS) sweetened with isomalt/stevia. Both snacks showed high bioactive compounds retention (>75%) and no significant changes in several physicochemical properties after 6 months storage. Fresh snacks were hard and crunchy, and the perception of consumers within liking categories was: HI: 43%, and IS: 72%. After storage HI snack showed higher acceptance by consumers (75% within liking categories) while IS snack showed a decrease in their acceptance (63% within disliking categories). The penalty analysis showed that the sensory shelf-life of both snacks would be limited by the changes produced in texture and color during storage. HI snack could reach six storage months, while IS would be stored up to 3 months. The use of diverse sweeteners allowed obtaining two different products which could be incorporated into a healthy diet.

Sensory characterization, acceptance, and stability studies on low calories fruit jelly candies.

The objective of this work was to characterize and evaluate the sensory acceptance and stability of jelly candies with flavor of orange (OJC) or raspberry (RJC), and alternative sweeteners (isomalt, maltitol syrup, and stevia). A qualitative sensory study showed that among potential buyers (70% of 200 participants), the main associations were related to taste, sensations/feelings, health, and appearance. A sensory panel (80 participants) performed the rating of liking, and the sensory intensity assays, which showed that both candies were rated within liking categories by more than 70% of consumers, and that the taste attributes were categorized at the right point. Color and gumminess were rated in the just point for RJC, while for OJC, these variables deviated from the expected. Regarding sensory stability, a penalty study advised that bitterness/sweetness ratio, and color attributes should be improved in order to extend the candies´ sensory shelf life. Additionally, a trained sensory panel evaluated the textural changes during storage of the jelly candies. The sensory studies carried out on the novel jelly candies showed substantial interest from consumers, therefore, they could be considered an alternative to replace traditional sweets, offering a nutritional contribution from fruits, without artificial preservatives and added free sugars. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05747-7.

Different methods for textural evaluation of freeze-dried candies during storage.

The textural changes during storage of two freeze-dried candies developed from blackcurrant fruits, unflavored yogurt, and different alternative sweeteners, one sweetened with honey/isomalt (HI) and another sweetened with isomalt/stevia (IS), were analyzed using three different methods (instrumental, sensory, and image analysis). Fresh candies were in the supercooled state and presented different structural and textural characteristics (HI: compact and homogeneous, and IS: porous and crunchy), with Fmax values of 139 ± 14 and 174 ± 16 N for HI and IS, respectively. After storage, the instrumental analysis showed approximately 60% average drop in Fmax and W values, in agreement with the decrease observed by sensory analysis in hardness, fracturability, and crispness. Image analysis showed an increase in parameters related to the homogeneity and the uniformity/smoothness for HI. Pearson's correlation coefficients analysis showed that there was a good correlation between the three techniques used, suggesting that the joint use of these methods could be performed for a better understanding of complex food texture.

Classification, quality characteristics, sensory perception and texture prediction of wooden breast myopathy in broilers from Argentina.

The objective of this study was to characterize the wooden breast (WB) myopathy in the poultry industry, and establish degrees of severity by analyzing the composition and important characteristics of meat quality. 175 chickens from the COBB-500 commercial line were analyzed, and three WB categories were established: normal, moderate, and severe. The prevalence was: 4.00% severe, 46.29% moderate, and 49.71% normal, and it was affected by sex and weight. The WB characterization was carried out on 7 breasts of each WB degree. A decrease in protein and ashes, an increase in fat and loss of water by dripping, and color changes were observed as the degree of severity advanced. The texture was evaluated by instrumental, sensory, and image analysis techniques. Severe samples showed alterations in compression test, cohesiveness and juiciness, together with significant differences on the parameters, "contrast" and "energy". A characterization of WB myopathy was achieved for the first time in Argentina. The decline in meat quality could lead to industrial losses. Image analysis proved to be a promising technique for differentiating the severity of WB myopathy in raw chicken. Differences between raw and cooked samples were detected, thus both types of meat should be studied in detail. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05608-9.

Colorant and antioxidant properties of freeze-dried extracts from wild berries: use of ultrasound-assisted extraction method and drivers of liking of colored yogurts.

This work aimed at developing powders rich in antioxidants and pigments from two wild berries: maqui (Aristotelia chilensis) and murra (Rubus ulmifolius). Fruits were subjected to successive ultrasound-assisted extractions (UAE) and then freeze-dried. Physical properties, anthocyanin stability of powders, and their performance as natural colorants in yogurts were evaluated. The optimum extraction methods were: UAE for 10 min in murra, and without UAE (control) in maqui, with juice extraction yields ranging between 80 and 82%. Maqui powder exhibited ≈ 2.8 times more polyphenol and anthocyanin content than murra. However, murra powder showed better stability characteristics as powder colorant since it exhibited greater protection of anthocyanins by means of copigmentation phenomena. Regarding consumer's perception of colored yogurt, samples with 4% and 8% maqui powder could be considered as future prototypes to be launched into the market. The obtained powders may be used in different industrial food applications.

Development of healthy gummy jellies containing honey and propolis.

BACKGROUND: The objective of this work was to develop gummy jellies containing honey and propolis, keeping the natural antioxidant principles of the beehive ingredients and satisfying consumer requirements. RESULTS: A gummy jelly containing honey and propolis (HPGJ) was developed. A sensory study with consumers (n = 74) performed an intensity level evaluation test analyzing color, hardness, adhesiveness, gumminess, sweet taste and honey taste. A penalty analysis indicated that HPGJ was too hard. However, in the global acceptance study, more than 90% of consumers gave liking categories. Regarding the potential functional properties, the antioxidant capacity (AC) of HPGJ was 8.17 ± 0.55 mmol Trolox equivalent kg-1 , and up to 40% AC was retained after in vitro digestion. Additionally, AC of HPGJ was up to ten times higher than that of similar commercial products. A storage study at 25 °C showed that color and AC increased significantly over 90 days owing to the development of Maillard reaction. Storage under darkness allowed keeping low values of global color change for up to 45 days. Another positive facet was that the addition of propolis delayed fungal growth during storage. CONCLUSION: An organoleptically palatable gummy jelly was obtained. Among the positive features, it showed higher AC than similar commercial candies. Additionally, HPGJ offered a high bioaccesible AC input detected upon in vitro digestion. Overall, HPGJ could be considered an interesting, appetizing and healthier alternative to regular gummy jellies available in the market. Adequate packaging should be considered in order to extend HPGJ shelf life, reducing browning reactions. © 2019 Society of Chemical Industry.

Valorization of postharvest sweet cherry discard for the development of dehydrated fruit ingredients: compositional, physical, and mechanical properties.

BACKGROUND: Sweet cherries are an excellent source of phenolic compounds, which may contribute to a healthy diet. The objective of this work was to generate dehydrated ingredients from postharvest discard of sweet cherries. RESULTS: Four dried ingredients were obtained from fresh sweet cherry discard (Lapins var.) using an osmotic dehydration pretreatment and freeze drying or air drying. The ingredients showed an important phenolic contribution (2.8-6.6 g gallic acid kg-1 of product) and preserved the natural color of the fruit to a great extent. Freeze-dried ingredients were less hygroscopic than air-dried ones, and presented with a softer texture. All the ingredients were in a supercooled state at room temperature (Tg range: -23.0 to -18.8 °C). Sugar infusion pretreatment caused a decrease in water sorption capacity and molecular mobility; it also reduced the initial rehydration rate. CONCLUSION: Relevant differences in nutritional and structural characteristics of the ingredients were observed depending on the processing method used. These ingredients could be incorporated into different processed foods, such as snacks, cereal mixtures, cereal bars, and bakery and confectionery products. Air-dried control ingredients presented better nutritional qualities and air-dried sweet cherries with sugar infusion pretreatment could be appropriate ingredients for applications where sweet flavor and slow rehydration rate are required. © 2018 Society of Chemical Industry.

Physical and functional properties of spray-dried powders from blackcurrant juice and extracts obtained from the waste of juice processing.

Blackcurrants contain high levels of polyphenolics, particularly flavonols and anthocyanins, which contribute to their high antioxidant activity. The aims of this work were the recovery of bioactive compounds from the remaining solid (waste) after processing blackcurrant juice and to obtain spray-dried powders from the blackcurrant juice and extracts. The extraction of bioactive compounds from the fruit pulp was performed by ultrasound-assisted extraction. Experiments were conducted to select the more suitable solvent, and citric acid was chosen. Then, to optimize the extraction conditions (time, solvent concentration, and amplitude) an experimental design using a Box-Behnken Design was done. Comparing the optimized extract with the fruit, 31% total monomeric anthocyanins, 19% total phenolic compounds, and 10% antioxidant capacity were obtained. The optimized extract and the juice were mixed and spray dried, using maltodextrin as carrier matrix. A blackcurrant powder with low hygroscopicity 14.46 ± 0.13 (g a.w./100 g d.m) and high solubility 94.25 ± 4% was obtained. High concentration of bioactive compounds and antioxidant capacity was recorded: Total monomeric anthocyanins 63.01 ± 1 (mg cyn-3-glu/100 g.d.m), total phenolic content 116.87 ± 5 (mg gallic acid/100 g d.m.), and antioxidant capacity 144.40 ± 0.11 (mg eq Trolox/100 g.d.m.).

Effect of Galacto-Oligosaccharides: Maltodextrin Matrices on the Recovery of Lactobacillus plantarum after Spray-Drying.

In this work maltodextrins were added to commercial galacto-oligosaccharides (GOS) in a 1:1 ratio and their thermophysical characteristics were analyzed. GOS:MD solutions were then used as matrices during spray-drying of Lactobacillus plantarum CIDCA 83114. The obtained powders were equilibrated at different relative humidities (RH) and stored at 5 and 20°C for 12 weeks, or at 30°C for 6 weeks. The Tgs of GOS:MD matrices were about 20-30°C higher than those of GOS at RH within 11 and 52%. A linear relation between the spin-spin relaxation time (T2) and T-Tg parameter was observed for GOS:MD matrices equilibrated at 11, 22, 33, and 44% RH at 5, 20, and 30°C. Spray-drying of L. plantarum CIDCA 83114 in GOS:MD matrices allowed the recovery of 93% microorganisms. In contrast, only 64% microorganisms were recovered when no GOS were included in the dehydration medium. Survival of L. plantarum CIDCA 83114 during storage showed the best performance for bacteria stored at 5°C. In a further step, the slopes of the linear regressions provided information about the rate of microbial inactivation for each storage condition (k values). This information can be useful to calculate the shelf-life of spray-dried starters stored at different temperatures and RH. Using GOS:MD matrices as a dehydration medium enhanced the recovery of L. plantarum CIDCA 83114 after spray-drying. This strategy allowed for the first time the spray-drying stabilization of a potentially probiotic strain in the presence of GOS.

Role of mono- and oligosaccharides from FOS as stabilizing agents during freeze-drying and storage of Lactobacillus delbrueckii subsp. bulgaricus.

The aim of this work was to assess the role of mono- and oligosaccharides present in fructo-oligosaccharides (FOS) mixtures as protective agents during freeze-drying and storage of Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333. Different FOS mixtures were enzymatically obtained from sucrose and further purified by removing the monosaccharides produced as secondary products. Their glass transition temperatures (Tg) were determined at 11, 22 and 33% relative humidity (RH). Bacterial cultures were freeze-dried in the presence of 20% w/v solutions of the studied FOS. Their protective effect during freeze-drying was assessed by bacterial plate counting, and by determining the lag time from growth kinetics and the uptake of propidium iodide (PI). Plate counting during bacterial storage at 4°C, and 11, 22 and 33% RH for 80days completed this rational analysis of the protective effect of FOS. Purification of FOS led to an increase of Tg in all the conditions assayed. Microorganisms freeze-dried in the presence of non-purified FOS were those with the shortest lag times. Bacteria freeze-dried with pure or commercial FOS (92% of total FOS) showed larger lag times (8.9-12.6h). The cultivability of microorganisms freeze-dried with non-purified FOS and with sucrose was not significantly different from that of bacteria before freeze-drying (8.74±0.14logCFU/mL). Pure or commercial FOS were less efficient in protecting bacteria during freeze-drying. All the protectants prevented membrane damage. The cultivability of bacteria freeze-dried with FOS decayed <1logarithmicunit after 80days of storage at 11% RH. When storing at 22 and 33% RH, pure and commercial FOS were those that best protected bacteria, and FOS containing monosaccharides were less efficient. The effect of FOS on bacterial protection is the result of a balance between monosaccharides, sucrose and larger FOS in the mixtures: the smallest sugars are more efficient in protecting lipid membranes, and the larger ones favor the formation of vitreous states.

Effect of physical properties on the stability of Lactobacillus bulgaricus in a freeze-dried galacto-oligosaccharides matrix.

The ability of galacto-oligosaccharides (GOS) to protect Lactobacillus delbrueckii subsp. bulgaricus upon freeze drying was analyzed on the basis of their capacity to form glassy structures. Glass transition temperatures (T(g)) of a GOS matrix at various relative humidities (RH) were determined by DSC. Survival of L. bulgaricus in a glassy GOS matrix was investigated after freezing, freeze drying, equilibration at different RHs and storage at different temperatures. At 32 °C, a drastic viability loss was observed. At 20 °C, the survival was affected by the water content, having the samples stored at lower RHs, the highest survival percentages. At 4°C, no decay in the cells count was observed after 45 days of storage. The correlation between molecular mobility [as measured by Proton nuclear magnetic resonance (¹H NMR)] and loss of viability explained the efficiency of GOS as cryoprotectants. The preservation of microorganisms was improved at low molecular mobility and this condition was obtained at low water contents and low storage temperatures. These results are important in the developing of new functional foods containing pre and probiotics.

Effect of trehalose on the interaction of Alzheimer's Aß-peptide and anionic lipid monolayers.

The interaction of amyloid ß-peptide (Aß) with cell membranes is believed to play a central role in the pathogenesis of Alzheimer's disease. In particular, recent experimental evidence indicates that bilayer and monolayer membranes accelerate the aggregation and amyloid fibril formation rate of Aß. Understanding that interaction could help develop therapeutic strategies for treatment of the disease. Trehalose, a disaccharide of glucose, has been shown to be effective in preventing the aggregation of numerous proteins. It has also been shown to delay the onset of certain amyloid-related diseases in a mouse model. Using Langmuir monolayers and molecular simulations of the corresponding system, we study several thermodynamic and kinetic aspects of the insertion of Aß peptide into DPPG monolayers in water and trehalose subphases. In the water subphase, the insertion of the Aß peptide into the monolayer exhibits a lag time which decreases with increasing temperature of the subphase. In the presence of trehalose, the lag time is completely eliminated and peptide insertion is completed within a shorter time period compared to that observed in pure water. Molecular simulations show that more peptide is inserted into the monolayer in the water subphase, and that such insertion is deeper. The peptide at the monolayer interface orients itself parallel to the monolayer, while it inserts with an angle of 50° in the trehalose subphase. Simulations also show that trehalose reduces the conformational change that the peptide undergoes when it inserts into the monolayer. This observation helps explain the experimentally observed elimination of the lag time by trehalose and the temperature dependence of the lag time in the water subphase.

Glass transition and time-dependent crystallization behavior of dehydration bioprotectant sugars.

It has been suggested that the crystallization of a sugar hydrate can provide additional desiccation by removing water from the amorphous phase, thereby increasing the glass transition temperature (T(g)). However, present experiments demonstrated that in single sugar systems, if relative humidity is enough for sugar crystallization, the amorphous phase will have a short life. In the conditions of the present experiments, more than 75% of amorphous phase crystallized in less than one month. The good performance of sugars that form hydrated crystals (trehalose and raffinose) as bioprotectants in dehydrated systems is related to the high amount of water needed to form crystals, but not to the decreased water content or increased T(g) of the amorphous phase. The latter effect is only temporary, and presumably shorter than the expected shelf life of pharmaceuticals or food ingredients, and is related to thermodynamic reasons: if there is enough water for the crystal to form, it will readily form.

Non-enzymatic browning kinetics analysed through water-solids interactions and water mobility in dehydrated potato.

Non-enzymatic browning (NEB) development was studied in dehydrated potato at 70°C. It was related to the macroscopic and molecular properties and to water-solid interactions over a wide range of water activities. Time resolved (1)H NMR, thermal transitions and water sorption isotherms were evaluated. Although non-enzymatic browning could be detected in the glassy state; colour development was higher in the supercooled state. The reaction rate increased up to a water content of 26g/100g of solids (aw=0.84) and then decreased at higher water contents, concomitantly with the increase of water proton mobility. The joint analyses of NEB kinetics, water sorption isotherm and proton relaxation behaviour made it evident that the point at which the reaction rate decreased, after a maximum value, could be related to the appearance of highly mobile water. The results obtained in this work indicate that the prediction of chemical reaction kinetics can be performed through the integrated analysis of water sorption, water and solids mobility and the physical state of the matrix.

Effects of trehalose on the phase behavior of DPPC-cholesterol unilamellar vesicles.

A systematic study is presented of the effects of trehalose on the physical properties of extruded DPPC-cholesterol unilamellar vesicles. Particular emphasis is placed on examining how the interactions present in the hydrated state translate into those in the dehydrated state. Observations from HSDSC and DSC are used to examine the phase behavior of hydrated and dehydrated vesicles, respectively. The concentration of trehalose inside and outside the vesicles is manipulated, and is shown to affect the relative stability of the membranes. Our results show for the first time that a combination of high inner and low outer trehalose concentration is able to decrease the gel-to-liquid crystalline phase temperature (T(m)), while any other combination will not. Upon dehydration, the T(m) of all lipid mixtures increases. The extent of the increase depends on the trehalose distribution across the bilayer. The T(m) changes in the same direction with trehalose concentration for both freeze-dried and fully hydrated samples, suggesting that the trehalose distribution across the vesicle membrane, as well as the trehalose-phospholipid interaction, is maintained upon lyophilization. The results presented in this work may aid in the formulation of systems to be used in the lyophilization of liposomes for drug delivery applications.

Inhibition of trehalose crystallization by cytoplasmic yeast components.

The influence of different yeast (Saccharomyces cerevisiae) cellular fractions was studied in an attempt to gain knowledge on the feasibility of trehalose crystallization in yeast cells. Certain constituents of S. cerevisiae cells inhibited/delayed trehalose crystallization upon humidification at high relative humidities.

Phase behavior of freeze-dried phospholipid-cholesterol mixtures stabilized with trehalose.

A study is presented of the role of cholesterol content on the gel-to-liquid crystalline phase transition of freeze-dried liposomes stabilized with trehalose, a well known lyoprotectant. The phospholipids considered in this work, DPPC and DPPE, belong to the two predominant phospholipid species found in numerous biological membranes. Cholesterol is found in abundance in mammalian plasma membranes. DSC measurements reveal that cholesterol-containing liposomes exhibit multiple phase transitions upon dehydration. Addition of trehalose to these systems lowers the phase transition temperature and limits the phase separation of the lipidic components upon freeze-drying. This work provides strong evidence for the effectiveness of trehalose in stabilizing cholesterol-containing membranes upon lyophilization.

Effect of pH, counter ion, and phosphate concentration on the glass transition temperature of freeze-dried sugar-phosphate mixtures.

PURPOSE: The aim of the present work is to study the interaction of phosphate salts with trehalose and sucrose in freeze-dried matrices, particularly the effect of the salts on the glass transition temperature (Tg) of the sugars. METHODS: Freeze-dried trehalose and sucrose systems containing different amounts of sodium or potassium phosphate were analyzed by differential scanning calorimetry to determine the Tg and by Fourier-transform infrared spectroscopy (FTIR) analysis to evaluate the strength of the interaction between sugars and phosphate ions. RESULTS: Sucrose-phosphate mixtures show an increase in Tg up to 40 degrees C in a broad pH range (4-9) compared to that of pure sucrose. Sucrose-phosphate mixtures exhibit a higher Tg than pure sucrose while retaining higher water contents. Trehalose-phosphate mixtures (having a Tg of 135 degrees C at a pH of 8.8) are a better option than pure trehalose for preservation of labile materials. The -OH stretching of the sugars in the presence of phosphates decreases with increase in pH, indicating an increase in the sugar-phosphate interaction. CONCLUSIONS: Sugar-phosphate mixtures exhibit several interesting features that make them useful for lyophilization of labile molecules; Tg values much higher than those observed for the pure sugars can be obtained upon the addition of phosphate.