Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
  • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
  • A23C20/00—Cheese substitutes
  • A23C20/02—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates
  • A23C20/025—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates mainly containing proteins from pulses or oilseeds
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
  • A23L11/30—Removing undesirable substances, e.g. bitter substances
  • A23L11/37—Removing undesirable substances, e.g. bitter substances using microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
  • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
  • A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms

Definitions

• the present disclosure generally relates to fermented plant-based products and methods of producing them through fermentation.
• dairy cheese is an ancient practice that started as a way of preserving milk and turned into the development of fermented products with a broad range of flavors and interesting textures that are nowadays widely consumed on a regular basis.
• the characteristic organoleptic properties of cheese are directly linked to the production processes, the performance of different microorganisms, and most importantly, the nature of dairy milk.
• This colloidal dispersion of fat globules stabilized by casein micelles presents extremely particular behavior upon heat treatment or acidification, and its replication with plant raw materials a difficult challenge to overcome.
• milk is heat-treated to ensure safety and inactivate endogenous microorganisms that could be present in the raw product.
• proteins denature and unfold.
• Microorganisms e.g. lactic acid bacteria
• This pH drop complements the action of rennet, encouraging proteins to interact with each other and start forming a protein network known as curd, that entraps fat globules in its pores and conforms the firm texture of cheese.
• Milk casein is responsible for the formation of this three-dimensional network, and its characteristic molecular structure is responsible for its versatility to confer liquid but also gel-like textures. For these reasons, knowledge on plant protein behavior under different processing conditions is required to develop high quality products.
• This fat is preferred because of its high melting point, which makes it remain solid at ambient temperature and therefore, provide firmer textures. Furthermore, it can melt when the temperature increases, namely in the mouth or during cooking. In addition, when adding agents such as carrageenans and other hydrocolloids, it is expected to obtain gel-like structures when including them in a liquid plant protein matrix that undergoes hydration and further heat treatment. The behavior of these ingredients is already well known, and still the textural properties of most plant-based cheeses are not fully satisfactory.
• the flavor profile of the currently commercialized plant-based cheeses is achieved by cheese flavoring agents or by the characteristic flavor of the plant raw material (Short et al.,
• the latter one implies the presence of beany, nutty, or earthy aftertaste with low acceptance levels among consumers.
• the potential of fermentation in flavor development is double: it can reduce the off-flavor from the raw material and it can boost the intensity of acid and dairy like notes reminiscent of animal milk products.
• the flavor profile of dairy cheese is formed by a complex mixture of very diverse volatile organic compounds (VOC) and it depends on factors such as VOC and precursors already present in milk and the metabolism of the bacteria that are fermenting it, among others. Therefore, replicating such a complex VOC mixture with different starting raw materials is a challenge since the precursors of the typical VOC of dairy cheese might not be found in plant protein matrices. VOC products of the carbohydrate metabolism are easier to obtain through plant protein matrices.
• VOC products of protein metabolism are produced after protein hydrolysis occurring during cheese maturation, and, as well as VOC products of fat metabolism, they are directly linked to the nature of those proteins and lipids that are present in animal milk.
• the present disclosure provides different blends of bacteria starter cultures and optionally bacteria adjunct cultures for texture development, reduction of beany flavor, and production of dairy-like volatile organic compounds in plant-based protein matrices derived from pea, and method of making foodstuff by fermentation.
• a method for producing a fermented plant-based product comprising a) adding a starter culture comprising Streptococcus thermophilus and Lactobacillus bulgaricus to a plant base, b) optionally adding an adjunct culture comprising Lactobacillus helveticus to the plant base, c) fermenting the plant base for a period of time until a target pH is reached, thereby producing the fermented plantbased product; wherein the plant base is a pea base.
• the starter culture further comprises Lactobacillus acidophilus, Lactobacillus paracasei and Bifidobacterium.
• the adjunct culture further comprises Pediococcus and Lactobacillus plantarum.
• the adjunct culture is added at the start of the fermentation period.
• the plant base is a liquid base with plant protein content of 5-15% (w/w), such as 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 % (w/w).
• the plant protein content is 10 % (w/w).
• a plant-based product obtainable by the method according to the first aspect is provided.
• a fermented plant-based product which has a pea protein content of 5-15% (w/w), such as 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 % (w/w) or higher than 15% and which comprises Streptococcus thermophilus and Lactobacillus bulgaricus, and optionally Lactobacillus helveticus.
• the fermented plant-based product further comprises Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus paracasei.
• the plant-based product further comprises Pediococcus and Lactobacillus plantarum.
• VegaTM, NU-TRISH®, L.CASEI 431®, and LGG® are trademarks of Chr. Hansen A/S.
• VegaTM Premium, VegaTM Vibe, VegaTM Harmony, VegaTM Classic, VegaTM Boost HA, VegaTM NU-TRISH®, VegaTM Boost PA, VegaTM NU-TRISH® L.CASEI 431®, VegaTM Boost LP and LGG® are well defined, quality controlled cultures, readily available as commercial products from Chr. Hansen A/S.
• starter culture is a culture which is a preparation (composition) of one or more bacterial strains (such as lactic acid bacteria strains) to assist the beginning of the fermentation process in preparation of fermented products such as various foods, feeds and beverages.
• adjunct culture is a culture which is a preparation (composition) of one or more bacterial strains (such as lactic acid bacteria strains) to further assist the fermentation process in preparation of fermented products such as various foods, feeds and beverages.
• lactic acid bacteria designates food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram positive, low-GC, acid tolerant, non-sporulating, non-respiring, rod-shaped bacilli or cocci. During the fermentation stage, the consumption of carbohydrate by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. These bacteria are thus generally responsible for the acidification of milk and for the texture of various dairy products.
• the industrially most useful lactic acid bacteria are found within the order "Lactobacillales" which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp. and Propionibacterium spp. These are frequently used as food cultures alone or in combination with other lactic acid bacteria.
• milk or ’’dairy is to be understood as the lacteal secretion obtained by milking any mammal, such as cows, sheep, goats, buffaloes or camels.
• the milk is cow's milk.
• milk also includes protein/fat solutions made partly or exclusively of plant materials.
• dairy analogue or “plant-based” product as used herein is meant to refers to dairy-like products, which are products used as culinary replacements for dairy products, prepared where one or more milk constituents have been replaced with other ingredients and the resulting food resembles the original product.
• the milk constituents are replaced completely or substantially with plant material, for example, using planted-based milks derived from legumes (such as soybeans, pea, lentils or chickpeas), nuts (such as coconut), cereals (such as oat).
• plant base is used to describe the plant material used as a base for fermentation.
• Figure 1 is a graph showing acidification of fermented Pea Protein Isolate (PPI) gels with different bacterial blends and their acidification slopes.
• PPI Pea Protein Isolate
• Figure 2 is a graph showing gel firmness of samples fermented with the same culture/adjunct combinations. Results are displayed as force values (g).
• Figure 3 shows spectra of volatile compounds related to beany flavor detected in fermented PPI samples whose production was significantly affected by the different bacterial blends. Results are displayed as signal-to-noise (S/N).
• Figure 4 shows spectra of Volatile compounds found in dairy cheeses and also detected in fermented PPI samples whose production was significantly affected by the different bacterial blends. Results are displayed as S/N. DETAILED DESCRIPTION OF THE INVENTION
• This disclosure relates to plant-based fermented foodstuff, and in particular plant-based cheese analog products.
• Texture and flavor development in plant-based cheese analog products generally relies on the use of texturizing and flavoring agents.
• fermentation can be a powerful tool to improve those sensorial attributes.
• Acidification performance of bacteria used for fermentation is an important criterion to determine whether they are commercially favorable or not. Furthermore, how the bacteria contribute to gel hardness of the plant base, and their ability to mask off-flavors and develop dairy-like flavors are important advantages, which can be monitored through pH measurement, compression tests, and volatile compounds analysis, respectively.
• PPI Pea protein isolate
• ADM American Type Culture Collection
• sunflower oil Oil
• sucrose sucrose
• dextrose Sigma Aldrich, S0borg, Denmark
• All bacterial cultures used in this example are from Chr. Hansen A/S (H0rsholm, Denmark). Table 1 shows an overview of the strains included in each culture and each adjunct combination.
• the pea protein isolate (PPI) matrix was prepared as previously described ((Masia, Jensen, Petersen and Buldo, 2022)). 10% pea protein isolate was suspended in a 1 % glucose and 1% sucrose water solution at 8,100 rpm with a L5M Laboratory Mixer (Silverson, Chesham,
• the protein suspension was emulsified with 10% sunflower oil under the same mixing conditions and homogenized with high pressure in a GEA Lab Homogenizer PandaPLUS 2000 (GEA, Parma, Italy) at two stages (150; 50 bars) in one pass.
• the matrix was pasteurized at 90°C for 20 minutes and cooled down to 43°C for further inoculation.
• Texture analysis The texture of the fermented gels was analyzed after 7 days of storage under refrigeration with a compression test in a Texture Analyzer (Stable Micro Systems, Surrey, United Kingdom). Each sample was cut in cylinder shape of 2 cm x 2 cm and compressed 5 mm with a plate geometry of 40 mm using a 5 kg load cell at 1 mm/s. The trigger was set to 15 g and the data acquisition rate was 500 pps. No oil was applied on the probe or in the sample prior to compression.
• the volatile compounds produced after fermentation and 7 days of storage under refrigeration were analyzed by head space solid phase microextraction gas chromatography coupled to mass spectrometry (HS-SPME-GC-MS).
• the instrument was a Multi Purpose Sampler (Gerstel, MSCI, Skovlunde, Denmark), with a 7890B GC (Agilent Technologies, Denmark) and a 5977A MS (Agilent Technologies, Denmark).
• VOCs were extracted by SPME using a DVB/Car/PDMS-fiber (Supelco#57299, VWR, Denmark) for 20 min.
• Oven temperature program was as follows: starting at 32°C/2min - increased to 102°C@10°C/min - further increased to 145°C@5°C/min - further increased to 200°C@15°C/min - further increased to 200°C@15°C/min - further increased to 280°C@20°C/min - hold at 280°C for 5 min.
• the mass spectrometer operated in electron impact mode at -70eV and the analyzer was scanning from 29-209 amu.
• NIST 17 library search and Retention Indexes were used for identification of VOCs. Feature extraction was done using MassHunter Quantitative Analysis (Version 10.2, Build 10.2.733.8, Agilent Technologies, Denmark) and results calculated as peak height divided by baseline noise (signal-to-noise, S/N). Samples were analyzed in biological duplicates after fermenting 3 grams of pea protein matrix directly in a 20 ml headspace vial. A removal ratio was calculated for the beany off-flavors comparing the detected S/N values of those in the fermented samples to those in the non-fermented PPI matrix.
• Acidification, gel firmness, and chromatography results were analyzed with an analysis of variance with a factorial to degree design, where the effect of the starter cultures, the effect of the adjuncts cultures combinations and the interaction between both were evaluated. Tukey tests were performed to define homogeneous subsets showing significance.
• the software used for the statistical analysis was JMP Pro 16 (SAS Institute, Cary, North Carolina, United States), and p values of less than 0.05 were interpreted as significant differences. All texture analysis were performed in biological triplicates and the analysis of volatile compounds was performed in duplicates.
• adjunct combinations samples without any adjunct cultures, namely those fermented just with starter cultures, showed significantly faster acidification than samples fermented with adjunct combination C4 (L helveticus, Pediococcus, L plantarum, and L casei).
• adjuncts have a slower acidification capacity and since they would consume part of the substrate that is available for the strains present in the starter culture, this one has less substrate to grow and produce more acid, causing slower pH drops.
• fast acidification was prioritized, since plant-based raw materials are microbially active and it is important to ensure safety by avoiding the growth of undesired microorganisms.
• blends including VegaTM Harmony and VegaTM Classic would be considered as acidification leads in this example.
• adjunct combinations CO and C1 belong to the same homogeneous group, according to the Tukey test results. This shows that the adjunct cultures in C1 , namely L helveticus and L casei, did not improve gel hardness when supplemented together and in absence of other adjunct cultures. Samples fermented with combination C3 were firmer than those fermented with C4 and those with C5. All three combinations share L helveticus, Pediococcus, and L plantarum, but C4 and C5 also contain L casei, or L casei with L rhamnosus, respectively. For a better understanding, the effect of the single strains present in each adjunct combination was evaluated.
• Pediococcus did not show any significant effect on gel hardness, which is also reflected in C1 and C2 not being significantly different.
• L rhamnosus did not show significant effects on gel firmness.
• C3 vs C4 it is possible to observe that the presence of L casei weakens the gel. From these results, it is possible to conclude that the presence of L plantarum and L casei would potentially reduce the firmness of the fermentation-induced gels produced in this example.
• L helveticus previously showed texturizing abilities in yogurt by producing exopolysaccharides (EPS) that interact with proteins and fill the pores in a yogurt gel.
• EPS exopolysaccharides
• this example focuses on cheese-like texture, and for instance, it has been reported that cheeses fermented with EPS-producing cultures presented lower hardness and consistency, among other textural parameters (Ahmed, El Soda, Hassan and Frank, 2005; Awad, Hassan and Muthukumarappan, 2005).
• fresh and aged dairy cheeses containing EPS producing strains previously presented less compact protein matrices (Dabour, Kheadr, Benhamou, Fliss and LaPointe, 2006).
• the VOC analysis with SPME-GC-MS detected 60 volatile compounds, from which 27 were selected for their contribution to beany flavor and cheese flavor for further statistical analysis. This selection was based on internal knowledge on off-flavor removal, especially green and beany notes, and formation of dairy notes compounds such as cheesy and buttery, as well as on existing studies on volatile compounds profile of fermented and unfermented pea matrices and dairy cheese products (Fischer, Cayot and Cachon, 2022; Ben-Harb et al., 2019a; Morales, Feliu and Ferna, 2004; Youssef et al., 2020)). These selected compounds comprised aldehydes, ketones, esters, furans, and sulfur derivative compounds.
• beany flavor results from a combination of different sensorial attributes, as combination of mold, earthy, green, and fresh pea (Fischer et al., 2022).
• the beany, green and/or grassy flavor of pea protein is mostly characterized by the aldehydes hexanal, 2- hexenal, heptanal, 2-heptenal, octanal, 2-octenal, pentanal, and 2,4-decadienal (Ben-Harb et al., 2019a; Trikusuma, Paravisini and Peterson, 2020a). Therefore, their degradation through fermentation is of great interest for plant-based cheese production.
• VegaTM Classic and VegaTM Harmony removed significantly greater levels of all these beany compounds, except for 2,4-decadienal, where no significant differences were observed with different blends, and these results are shown in Fig 3.
• Increasing levels of 2,4-decadienal upon UHT treatment of pea beverages were previously reported and related it to the oxidation of linoleic acid, the main fatty acid in pea (Trikusuma, Paravisini and Peterson, 2020b). This might have occurred also in the samples fermented in this example, and potentially due to further oxidation of the lipids in the sunflower oil.
• the blend containing VegaTM Classic + C5 was the one that degraded the highest levels of hexanal with a 73% removal ratio, whereas VegaTM Premium without adjuncts was the least efficient with 24% removal ratio. Furthermore, this latter blend degraded significantly less hexanal than any combination containing VegaTM Harmony and VegaTM Classic. In the case of heptanal, octanal, and pentanal, also VegaTM Classic + C5 was the most efficient with 76%, 78%, and 70% removal ratios, respectively. These results support the idea of combining the starter cultures with adjuncts to remove the off-flavors of legume raw materials such as pea.
• Blends containing VegaTM Harmony and VegaTM Classic as starter cultures, and furthermore blends containing adjunct combinations C5, C3, or C4 would be ideal to reduce the beany flavor in pea protein gels.
• 3-methylbutanal, an aldehyde derived from isoleucine and leucine providing malty and nutty notes (Utz, Spaccasassi, Kreissl, Stark, Tanger, Kulozik, Hofmann and Dawid, 2022; Ben-Harb, Saint-Eve, Panouille, Souchon, Bonnarme, Dugat-Bony and I rlinger, 2019b), was previously found in pea protein isolate (Utz et al., 2022).
• Benzaldehyde can be found in semi-hard cheeses (Molimard and Spinnler, 1996) and its production was associated to phenylalanine degradation in lactic acid bacteria (Nierop Groot and de Bont, 1998). In this example, its presence was only affected by the starter cultures. Samples fermented with VegaTM Premium and VegaTM Vibe presented significantly higher levels of this aldehyde in comparison to VegaTM Harmony and VegaTM Classic. Masja et al.
• Ketones were detected in all fermented PPI gels. These compounds are common in cheese, typically increasing during ripening, and bringing fruity-floral notes to the cheese as well as green, blue cheese (2-heptanone), and hot milk and musty (2-nonanone) aromas. Among them, 2,3-pentanedione was detected in all samples (Fig 4), with an increase of 95-99% in comparison to the unfermented base. It is a compound found in cheeses such as Mozzarella (Natrella, Faccia, Lorenzo, De Palo and Gambacorta, 2020) that can impart butter, creamy, nutty and cheese notes.
• Mozzarella Nazzarella, Faccia, Lorenzo, De Palo and Gambacorta, 2020
• Plant-dairy protein blends gelation behaviour in a filled particle matrix. Food Structure 29, 100198.
• Lactic acid fermentation A novel approach to eliminate unpleasant aroma in pea protein isolates. Lwt 150, 111927.
• Trikusuma M., Paravisini, L, Peterson, D.G., 2020b. Identification of aroma compounds in pea protein UHT beverages.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Food Science & Technology (AREA)
• Polymers & Plastics (AREA)
• Microbiology (AREA)
• Health & Medical Sciences (AREA)
• Nutrition Science (AREA)
• Agronomy & Crop Science (AREA)
• Botany (AREA)
• Biotechnology (AREA)
• Dairy Products (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=85239120

Family Applications (1)

Country Status (2)

Families Citing this family (1)

Family Cites Families (6)

• 2024
  • 2024-02-13 WO PCT/EP2024/053616 patent/WO2024170562A1/en not_active Ceased
  • 2024-02-13 EP EP24704211.2A patent/EP4665166A1/de active Pending

Also Published As

Similar Documents

Legal Events